Human sel-10 polypeptides and polynucleotides that encode them

ABSTRACT

The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding either of two alternative splice variants of human sel-10, one of which is expressed in hippocampal cells, and one of which is expressed in mammary cells. The invention also provides isolated sel-10 polypeptides and cell lines which express them in which Aβ processing is altered.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. Ser. No. 09/213,888,filed Dec. 17, 1998, which claims the benefit of the followingprovisional application: U.S. Serial No. 60/068,243, filed Dec. 19,1997, under 35 USC 119(e)(1).

FIELD OF THE INVENTION

[0002] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding either of two alternative splicevariants of human sel-10, one of which is expressed in hippocampalcells, and one of which is expressed in mammary cells. The inventionalso provides isolated sel-10 polypeptides.

BACKGROUND OF THE INVENTION

[0003] Alzheimer's disease (AD) is a degenerative disorder of thecentral nervous system which causes progressive memory and cognitivedecline during mid to late adult life. The disease is accompanied by awide range of neuropathologic features including extracellular amyloidplaques and intra-neuronal neurofibrillary tangles. (Sherringtol, R., etal.; Nature 375: 754-60 (1995)). Although the pathogenic pathway leadingto AD is not well understood, several genetic loci are known to beinvolved in the development of the disease.

[0004] Genes associated with early onset Alzheimer's disease (AD) havebeen identified by the use of mapping studies in families withearly-onset AD. These studies have shown that genetic loci onchromosomes 1 and 14 were likely to be involved in AD. Positionalcloning of the chromosome 14 locus identified a novel mutant geneencoding an eight-transmembrane domain protein which subsequently wasnamed presenilin-1 (PS-1). (Sherrington, R., et al.; Nature 375: 754-60(1995)). Blast search of the human EST database revealed a single ESTexhibiting homology to PS-1, designated presenilin-2 (PS-2) which wasshown to be the gene associated with AD on chromosome 1. (Levy-Lahad, E.et al., Science 269:973-977 (1995); Rogaev, E. I., et al., Nature 376:775-8 (1995); Li, J. et al., Proc. Natl. Acad. Sci. U.S.A. 92:12180-12184 (1995)). altered by the point mutations found in familialAlzheimer's disease [Perez-Tur, J. et al., Neuroreport 7: 297-301(1995); Mercken, M. et al., FEBS Lett. 389: 297-303 (1996)]. PS-1 geneexpression is widely distributed across tissues, while the highestlevels of PS-2 mRNA are found in pancreas and skeletal muscle. (Li, J.et al., Proc. Nat. Acad. Sci. U.S.A. 92: 12180-12184 (1995); Jinhe Li,personal communication). The highest levels of PS-2 protein, however,are found in brain (Jinhe Li, personal communication). Both PS-1 andPS-2 proteins have been localized to the endoplasmic reticulum, theGolgi apparatus, and the nuclear envelope. (Jinhe Li, personalcommunication; Kovacs, D. M. et al., Nat. Med. 2:224-229 (1996); Doan,A. et al., Neuron 17: 1023-1030 (1996)). Mutations in either the PS-1gene or the PS-2 gene alter the processing of the amyloid proteinprecursor (APP) such that the ratio of A-beta₁₋₄₂ is increased relativeto A-beta₁₋₄₀ (Scheuner, D. et al., Nat. Med. 2: 864-870 (1996)). Whencoexpressed in transgenic mice with human APP, a similar increase in theratio of A-beta₁₋₄₂ as compared to A-beta₁₋₄₀ is observed (Borchelt, D.R. et al., Neuron 17: 1005-1013 (1996); Citron, M. et al., Nat. Med. 3:67-72 (1997); Duff, K. et al., Nature 383: 710-713 (1996)), togetherwith an acceleration of the deposition of A-beta in amyloid plaques(Borchelt et al., Neuron 19: 939 (1997).

[0005] Despite the above-described observations made with respect to therole of PS-1 and PS-2 in AD, their biological function remains unknown,placing them alongside a large number of human disease genes having anunknown biological function. Where the function of a gene or its productis unknown, genetic analysis in model organisms can be useful in placingsuch genes in known biochemical or genetic pathways. This is done byscreening for extragenic mutations that either suppress or enhance theeffect of mutations in the gene under analysis. For example, extragenicsuppressors of loss-of-function mutations in a disease gene may turn onthe affected genetic or biochemical pathway downstream of the mutantgene, while suppressers of gain-of-function mutations will probably turnthe pathway off.

[0006] One model organism that can be used in the elucidation of thefunction of the presenilin genes is C. elegans, which contains threegenes having homology to PS-1 and PS-2, with sel-12 having the highestdegree of homology to the genes encoding the human presenifins. Sel-12was discovered in a screen for genetic suppressers of an activated notchreceptor, lin-12(d) (Levitan, D. et al., Nature 377: 351-354 (1995)).Lin-12 functions in development to pattern cell lineages. Hypermorphicmutations such as lin-12(d), which increase lin-12 activity, cause a“multi-vulval” phenotype, while hypomorphic mutations which decreaseactivity cause eversion of the vulva, as well as homeotic changes inseveral other cell lineages (Greenwald, I., et al., Nature 346: 197-199(1990); Sundaram, M. et al., Genetics 135: 755-763 (1993)). Sel-12mutations suppress hypermorphic lin-12(d) mutations, but only if thelin-12(d) mutations activate signaling by the intact lin-12(d) receptor(Levitan, D. et al., Nature 377: 351-354 (1995)). Lin-12 mutations thattruncate the cytoplasmic domain of the receptor also activate signaling(Greenwald, I., et al., Nature 346: 197-199 (1990)), but are notsuppressed by mutations of sel-12 (Levitan, D. et al., Nature 377:351-354 (1995)). This implies that sel-12 mutations act upstream of thelin-12 signaling pathway, perhaps by decreasing the amount of functionallin-12 receptor present in the plasma membrane. In addition tosuppressing certain lin-12 hypermorphic mutations, mutations to sel-12cause a loss-of-function for egg laying, and thus internal accumulationof eggs, although the mutants otherwise appear anatomically normal(Levitan, D. et al., Nature 377: 351-354 (1995)). Sel-12 mutants can berescued by either human PS-1 or PS-2, indicating that sel-12, PS-1 andPS-2 are functional homologues (Levitan, D., et al., Proc. Natl. Acad.Sci. U.S.A 93: 14940-14944 (1996)).

[0007] A second gene, sel-10, has been identified in a separate geneticscreen for suppressors of lin-12 hypomorphic mutations. Loss-of-functionmutations in sel-10 restore signaling by lin-12 hypomorphic mutants. Asthe lowering of sel-10 activity elevates lin-12 activity, it can beconcluded that sel-10 acts as a negative regulator of lin-12 signaling.Sel-10 also acts as a negative regulator of sel-12, the C. eleganspresenilin homologue (Levy-Lahad, E. et al., Science 269:973-977(1995)). Loss of sel-10 activity suppresses the egg laying defectassociated with hypomorphic mutations in sel-12 (Iva Greenwald, personalcommunication). The effect of loss-of-function mutations to sel-10 onlin-12 and sel-12 mutations indicates that sel-10 acts as a negativeregulator of both lin-12/notch and presenilin activity. Thus, a humanhomologue of C. elegans sel-10 would be expected to interact geneticallyand/or physiologically with human presenilin genes in ways relevant tothe pathogenesis of Alzheimer's Disease.

[0008] In view of the foregoing, it will be clear that there is acontinuing need for the identification of genes related to AD, and forthe development of assays for the identification of agents capable ofinterfering with the biological pathways that lead to AD.

INFORMATION DISCLOSURE

[0009] Hubbard E J A, Wu G, Kitajewski J, and Greenwald 1 (1997) Sel-10,a negative regulator of lin-12 activity in Caenorhabditis elegans,encodes a member of the CDC4 family of proteins. Genes & Dev11:3182-3193.

[0010] Greenwald-I; Seydoux-G (1990) Analysis of gain-of-functionmutations of the lin-12 gene of Caenorhabditis elegans. Nature. 346:197-9

[0011] Kim T-W, Pettingeli W H, Hallmark O G, Moir R D, Wasco W, Tanzi R(1997) Endoproteolytic cleavage and proteasomal degradation ofpresenilin 2 in transfected cells. J Biol Chem 272:11006-11010.

[0012] Levitan-D; Greenwald-I (1995) Facilitation of lin-12-mediatedsignalling by sel-12, a Caenorhabditis elegans S 182 Alzheimer's diseasegene. Nature. 377: 351-4.

[0013] Levitan-D; Doyle-T G; Brousseau-D; Lee-M K; Thinakaran-G; Slunt-HH; Sisodia-S S; Greenwald-I (1996) Assessment of normal and mutant humanpresenilin function in Caenorhabditis elegans. Proc. Natl. Acad. Sci.U.S.A. 93: 14940-4.

[0014] Sundaram-M; Greenwald-I (1993) Suppressors of a lin-12 hypomorphdefine genes that interact with both lin-12 and glp-1 in Caenorhabditiselegans. Genetics. 135: 765-83.

[0015] Sundaram-M; Greenwald-I (1993) Genetic and phenotypic studies ofhypomorphic lin-12 mutants in Caenorhabditis elegans. Genetics. 135:755-63.

[0016] F55B 12.3 GenPep Report (WMBL locus CEF55B 12, accession z79757).

SUMMARY OF THE INVENTION

[0017] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding human sel-10, which is expressed inhippocampal cells and in mammary cells. Unless otherwise noted, anyreference herein to sel-10 will be understood to refer to human sel-10,and to encompass both hippocampal and mammary sel-10. Fragments ofhippocampal sel-10 and mammary sel-10 are also provided.

[0018] In a preferred embodiment, the invention provides an isolatednucleic acid molecule comprising a polynucleotide having a sequence atleast 95% identical to a sequence selected from the group consisting of:

[0019] (a) a nucleotide sequence encoding a human sel-10 polypeptidehaving the complete amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, andSEQ ID NO:7, or as encoded by the cDNA clone contained in ATCC DepositNo.98978;

[0020] (b) a nucleotide sequence encoding a human sel-10 polypeptidehaving the complete amino acid sequence selected from the groupconsisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, or as encodedby the cDNA clone contained in ATCC Deposit No. 98979; and

[0021] (c) a nucleotide sequence complementary to the nucleotidesequence of (a) or (b).

[0022] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent conditions to a polynucleotide encoding sel-10, or fragmentsthereof.

[0023] The present invention also provides vectors comprising theisolated nucleic acid molecules of the invention, host cells into whichsuch vectors have been introduced, and recombinant methods of obtaininga sel-10 polypeptide comprising culturing the above-described host celland isolating the sel-10 polypeptide.

[0024] In another aspect, the invention provides isolated sel-10polypeptides, as well as fragments thereof. In a preferred embodiment,the sel-10 polypeptides have an amino acid sequence selected from thegroup consisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, and 10. Isolatedantibodies, both polyclonal and monoclonal, that bind specifically tosel-10 polypeptides are also provided.

BRIEF DESCRIPTION OF THE FIGURES

[0025] FIGS. 1A and 1B: FIGS. 1A and 1B are western blots showingprotein expression in HEK293 cells transfected with PS1-C-FLAG,6-myc-N-sel-10, and APP695NL-KK cDNAs.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding human sel-10. The nucleotidesequence of human hippocampal sel-10 (hhsel-10), which sequence is givenin SEQ ID NO:1, encodes five hhsel-10 polypeptides (hhsel-10-(1),hhsel-10-(2), hhsel-10-(3), hhsel-10-(4), and hhsel-10-(5), referred tocollectively herein as hhsel-10). The nucleotide sequence of humanmammary sel-10 (hmsel-10), which sequence is given in SEQ ID NO:2,encodes three hmsel-10 polypeptides (hmSel-10-(1), hmSel-10-(2), andhmsel-10-(3), referred to collectively herein as hmsel-10). Thenucleotide sequences of the hhsel-10 polynucleotides are given in SEQ IDNO. 1, where nucleotide residues 45-1928 of SEQ ID NO. 1 correspond tohhsel-110-(1), nucleotide residues 150-1928 of SEQ ID NO. 1 correspondto hhSel-10-(2), nucleotide residues 267-1928 of SEQ ID NO. 1 correspondto hhSel-10-(3), nucleotide residues 291-1928 of SEQ ID NO. 1 correspondto hhSel-10-(4), and nucleotide residues 306-1928 of SEQ ID NO. 1correspond to hhSel-10-(5). The nucleotide sequences of the hmSel-10polynucleotides are given in SEQ ID NO. 2, where nucleotide residues180-1949 of SEQ ID NO. 2 correspond to hmSel-10-(1), nucleotide residues270-1949 of SEQ ID NO. 2 correspond to hmSel-10-(2), and nucleotideresidues 327-1949 of SEQ ID NO. 2 correspond to hmSel-10-(3). The aminoacid sequences of the polypeptides encoded by the hhSel-10 and hm-Sel-10nucleic acid molecules are given as follows: SEQ ID NOS: 3, 4, 5, 6, and7 correspond to the hhSel-10-(1), hhSel-10-(2), hhSel-10-(3).hhSel-10-(4), and hhSel-10-(5) polypeptides, respectively, and SEQ IDNOS: 8, 9, and 10 correspond to the hmSel-10-(1), hmSel-10-(2), andhmSel-10-(3) polypeptides, respectively. Unless otherwise noted, anyreference herein to sel-10 will be understood to refer to human sel-10,and to encompass all of the hippocampal and mammary sel-10 nucleic acidmolecules (in the case of reference to sel-10 nucleic acid,polynucleotide, DNA, RNA, or gene) or polypeptides (in the case ofreference to sel-10 protein, polypeptide, amino acid sequnce). Fragmentsof hippocampal sel-10 and mammary sel-10 nucleic acid molecules andpolypeptides are also provided.

[0027] The nucleotide sequence of SEQ ID NO:1 was obtained as describedin Example 1, and is contained in cDNA clone PNV 102-1, which wasdeposited on Nov. 9, 1998, at the American Type Culture Collection,10801 University Blvd., Manassas, Va. 20110, and given accession number98978. The nucleotide sequence of SEQ ID NO:2 was obtained as describedin Example 1, and is contained in cDNA clone PNV 108-2, which wasdeposited on Nov. 9, 1998, at the American Type Culture Collection,10801 University Blvd., Manassas, Va. 20110, and given accession number98979.

[0028] The human sel-10 polypeptides of the invention share homologywith C. elegans sel-10, as well as with members of the β-transducinprotein family, including yeast CDC4, and human LIS-1. This familv ischaracterized by the presence of an F-box and multiple WD-40 repeats(Li, J., et al., Proc. Natl. Acad. Sci. U.S.A. 92:12180-12184 (1995)).The repeats are 20-40 amino acids long and are bounded by gly-his (GH)and trp-asp (WD) residues. The three dimensional structure ofβ-transducin indicates that the WD40 repeats form the arms of aseven-bladed propeller like structure (Sondek, J., et al., Nature379:369-374 (1996)). Each blade is formed by four alternating pleats ofbeta-sheet with a pair of the conserved aspartic acid residues in theprotein motif forming the limits of one internal beta strand. WD40repeats are found in over 27 different proteins which represent diversefunctional classes (Neer, E. J., et al., Nature 371:297-300 (1994)).These regulate cellular functions including cell division, cell fatedetermination, gene transcription, signal transduction, proteindegradation, mRNA modification and vesicle fusion. This diversity infunction has led to the hypothesis that β-transducin family membersprovide a common scaffolding upon which multiprotein complexes can beassembled.

[0029] The nucleotide sequence given in SEQ ID NO:1 corresponds to thenucleotide sequence encoding hhsel-10, while the nucleotide sequencegiven in SEQ ID NO:2 corresponds to the nucleotide sequence encodinghmsel-10. The isolation and sequencing of DNA encoding sel-10 isdescribed below in Examples 1 and 2.

[0030] As is described in Examples 1 and 2, automated sequencing methodswere used to obtain the nucleotide sequence of sel-10. The sel-10nucleotide sequences of the present invention were obtained for both DNAstrands, and are believed to be 100% accurate. However, as is known inthe art, nucleotide sequence obtained by such automated methods maycontain some errors. Nucleotide sequences determined by automation aretypically at least about 90%, more typically at least about 95% to atleast about 99.9% identical to the actual nucleotide sequence of a givennucleic acid molecule. The actual sequence may be more preciselydetermined using manual sequencing methods, which are well known in theart. An error in sequence which results in an insertion or deletion ofone or more nucleotides may result in a frame shift in translation suchthat the predicted amino acid sequence will differ from that which wouldbe predicted from the actual nucleotide sequence of the nucleic acidmolecule, starting at the point of the mutation. The sel-10 DNA of thepresent invention includes cDNA, chemically synthesized DNA, DNAisolated by PCR, genomic DNA, and combinations thereof. Genomic sel-10DNA may be obtained by screening a genomic library with the sel-10 cDNAdescribed herein, using methods that are well known in the art. RNAtranscribed from sel-10 DNA is also encompassed by the presentinvention.

[0031] Due to the degeneracy of the genetic code, two DNA sequences maydiffer and yet encode identical amino acid sequences. The presentinvention thus provides isolated nucleic acid molecules having apolynucleotide sequence encoding any of the sel-10 polypeptides of theinvention, wherein said polynucleotide sequence encodes a sel-10polypeptide having the complete amino acid sequence of SEQ ID NOs:3-10,or fragments thereof.

[0032] Also provided herein are purified sel-10 polypeptides, bothrecombinant and non-recombinant. Variants and derivatives of nativesel-10 proteins that retain any of the biological activities of sel-10are also within the scope of the present invention. As is describedabove, the sel-10 polypeptides of the present invention share homologywith yeast CDC4. As CDC4 is known to catalyze ubiquitination of specificcellular proteins (Feldman et al., Cell 91:221 (1997)), it may beinferred that sel-10 will also have this activity. Assay procedures fordemonstrating such activity are well known, and involve reconstitutionof the ubiquitinating system using purified human sel-10 proteintogether with the yeast proteins Cdc4p, Cdc53p and Skplp, or their humanorthologs, and an E1 enzyme, the E2 enzyme Cdc34p or its human ortholog,ubiquitin, a target protein and an ATP regenerating system (Feldman etal., 1997). Skplp associates with Cdc4p through a protein domain calledan F-box (Bai et al., Cell 86:263 (1996)). The F-box protein motif isfound in yeast CDC4, C. elegans sel-10, mouse sel-10 and human sel-10.The sel-10 ubiquitination system may be reconstituted with the C.elegans counterparts of the yeast components, e.g., cul-1 (also known aslin-19) protein substituting for Cdc53p (Kipreos et al., Cell 85:829(1996)) and the protein F46A9 substituting for Skplp, or with theirmammalian counterparts, e.g., Cu1-2 protein substituting for Cdc53p(Kipreos et al., 1996) and mammalian Skplp substituting for yeast Skplp.A phosphorylation system provided by a protein kinase is also includedin the assay system as per Feldman et al., 1997.

[0033] Sel-10 variants may be obtained by mutation of nativesel-10-encoding nucleotide sequences, for example. A sel-10 variant, asreferred to herein, is a polypeptide substantially homologous to anative sel-10 but which has an amino acid sequence different from thatof native sel-10 because of one or more deletions, insertions, orsubstitutions in the amino acid sequence. The variant amino acid ornucleotide sequence is preferably at least about 80% identical, morepreferably at least about 90% identical, and most preferably at leastabout 95% identical, to a native sel-10 sequence. Thus, a variantnucleotide sequence which contains, for example, 5 point mutations forevery one hundred nucleotides, as compared to a native sel-10 gene, willbe 95% identical to the native protein. The percentage of sequenceidentity, also termed homology, between a native and a variant sel-10sequence may also be determined, for example, by comparing the twosequences using any of the computer programs commonly employed for thispurpose, such as the Gap program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,Madison Wis.), which uses the algorithm of Smith and Waterman (Adv.Appl. Math. 2: 482-489 (1981)).

[0034] Alterations of the native amino acid sequence may be accomplishedby any of a number of known techniques. For example, mutations may beintroduced at particular locations by procedures well known to theskilled artisan, such as oligonucleotide-directed mutagenesis, which isdescribed by Walder et al. (Gene 42:133 (1986)); Bauer et al. (Gene37:73 (1985)); Craik (BioTechniques, January 1985, pp. 12-19); Smith etal. (Genetic Engineering: Principles and Methods, Plenum Press (1981));and U.S. Pat. Nos. 4,518,584 and 4,737,462.

[0035] Sel-10 variants within the scope of the invention may compriseconservatively substituted sequences, meaning that one or more aminoacid residues of a sel-10 polypeptide are replaced by different residuesthat do not alter the secondary and/or tertiary structure of the sel-10polypeptide. Such substitutions may include the replacement of an aminoacid by a residue having similar physicochemical properties, such assubstituting one aliphatic residue (Ile, Val, Leu or Ala) for another,or substitution between basic residues Lys and Arg, acidic residues Gluand Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, oraromatic residues Phe and Tyr. Further information regarding makingphenotypically silent amino acid exchanges may be found in Bowie et al.,Science 247:1306-1310 (1990). Other sel-10 variants which might retainsubstantially the biological activities of sel-10 are those where aminoacid substitutions have been made in areas outside functional regions ofthe protein.

[0036] In another aspect, the invention provides an isolated nucleicacid molecule comprising a polynucleotide which hybridizes understringent conditions to a portion of the nucleic acid moleculesdescribed above, e.g., to at least about i5 nucleotides, preferably toat least about 20 nucleotides, more preferably to at least about 30nucleotides, and still more preferably to at least about from 30 to atleast about 100 nucleotides, of one of the previously described nucleicacid molecules. Such portions of nucleic acid molecules having thedescribed lengths refer to, e.g., at least about 15 contiguousnucleotides of the reference nucleic acid molecule. By stringenthybridization conditions is intended overnight incubation at about 42/Cfor about 2.5 hours in 6×SSC/0.1% SDS, followed by washing of thefilters in 1.0×SSC at 65/C, 0.1% SDS.

[0037] Fragments of the sel-10-encoding nucleic acid molecules describedherein, as well as polynucleotides capable of hybridizing to suchnucleic acid molecules may be used as a probe or as primers in apolymerase chain reaction (PCR). Such probes may be used, e.g., todetect the presence of sel-10 nucleic acids in in vitro assays, as wellas in Southern and northern blots. Cell types expressing sel-10 may alsobe identified by the use of such probes. Such procedures are well known,and the skilled artisan will be able to choose a probe of a lengthsuitable to the particular application. For PCR, 5′ and 3′ primerscorresponding to the termini of a desired sel-10 nucleic acid moleculeare employed to isolate and amplify that sequence using conventionaltechniques.

[0038] Other useful fragments of the sel-10 nucleic acid molecules areantisense or sense oligonucleotides comprising a single-stranded nucleicacid sequence capable of binding to a target sel-10 mRNA (using a sensestrand), or sel-10 DNA (using an antisense strand) sequence.

[0039] In another aspect, the invention includes sel-10 polypeptideswith or without associated native pattern glycosylation. Sel-10expressed in yeast or mammalian expression systems (discussed below) maybe similar to or significantly different from a native sel-10polypeptide in molecular weight and glycosylation pattern. Expression ofsel-10 in bacterial expression systems will provide non-glycosylatedsel-10.

[0040] The polypeptides of the present invention are preferably providedin an isolated form, and preferably are substantially purified. Sel-10polypeptides may be recovered and purified from recombinant cellcultures by well-known methods, including ammonium sulfate or ethanolprecipitation, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography, hydroxylapatite chromatography and lectinchromatography. In a preferred embodiment, high performance liquidchromatography (HPLC) is employed for purification.

[0041] The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host celltransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. Because the invention also provides sel-10 polypeptides expressedfrom the polynucleotide molecules described above, vectors for theexpression of sel-10 are preferred. The vectors include DNA encoding anyof the sel-10 polypeptides described above or below, operably linked tosuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, mRNA ribosomal binding sites, and appropriatesequences which control transcription and translation. Nucleotidesequences are operably linked when the regulatory sequence functionallyrelates to the DNA encoding sel-10. Thus, a promoter nucleotide sequenceis operably linked to a sel-10 DNA sequence if the promoter nucleotidesequence directs the transcription of the sel-10 sequence.

[0042] Selection of suitable vectors to be used for the cloning ofpolynucleotide molecules encoding sel-10, or for the expression ofsel-10 polypeptides, will of course depend upon the host cell in whichthe vector will be transformed, and, where applicable, the host cellfrom which the sel-10 polypeptide is to be expressed. Suitable hostcells for expression of sel-10 polypeptides include prokaryotes, yeast,and higher eukaryotic cells, each of which is discussed below.

[0043] The sel-10 polypeptides to be expressed in such host cells mayalso be fusion proteins which include regions from heterologousproteins. Such regions may be included to allow, e.g., secretion,improved stability, or facilitated purification of the polypeptide. Forexample, a sequence encoding an appropriate signal peptide can beincorporated into expression vectors. A DNA sequence for a signalpeptide (secretory leader) may be fused in-frame to the sel-10 sequenceso that sel-10 is translated as a fusion protein comprising the signalpeptide. A signal peptide that is functional in the intended host cellpromotes extracellular secretion of the sel-10 polypeptide. Preferably,the signal sequence will be cleaved from the sel-10 polypeptide uponsecretion of sel-10 from the cell. Non-limiting examples of signalsequences that can be used in practicing the invention include the yeastI-factor and the honeybee melatin leader in sf9 insect cells.

[0044] In a preferred embodiment, the sel-10 polypeptide will be afusion protein which includes a heterologous region used to facilitatepurification of the polypeptide. Many of the available peptides used forsuch a function allow selective binding of the fusion protein to abinding partner. For example, the sel-10 polypeptide may be modified tocomprise a peptide to form a fusion protein which specifically binds toa binding partner, or peptide tag. Non-limiting examples of such peptidetags include the 6-His tag, thioredoxin tag, FLAG tag, hemaglutinin tag,GST tag, and OmpA signal sequence tag. As will be understood by one ofskill in the art, the binding partner which recognizes and binds to thepeptide may be any molecule or compound including metal ions (e.g.,metal affinity columns), antibodies, or fragments thereof, and anyprotein or peptide which binds the peptide. These tags may be recognizedby fluorescein or rhodamine labeled antibodies that react specificallywith each type of tag

[0045] Suitable host cells for expression of sel-10 polypeptides includeprokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotichosts to be used for the expression of sel-10 include bacteria of thegenera Escherichia, Bacillus, and Salmonella, as well as members of thegenera Pseudomonas, Streptomyces, and Staphylococcus. For expression in,e.g., E. coli, a sel-10 polypeptide may include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in aprokaryotic host. The N-terminal Met may optionally then be cleaved fromthe expressed sel-10 polypeptide.

[0046] Expression vectors for use in prokaryotic hosts generallycomprise one or more phenotypic selectable marker genes. Such genesgenerally encode, e.g., a protein that confers antibiotic resistance orthat supplies an auxotrophic requirement. A wide variety of such vectorsare readily available from commercial sources. Examples include pSPORTvectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, Md.),Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

[0047] Sel-10 may also be expressed in yeast host cells from generaincluding Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hostsare S. cerevisiae and P. pastoris. Yeast vectors will often contain anorigin of replication sequence from a 2T yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene.

[0048] Vectors replicable in both yeast and E. coli (termed shuttlevectors) may also be used. In addition to the above-mentioned featuresof yeast vectors, a shuttle vector will also include sequences forreplication and selection in E. coli. Direct secretion of sel-10polypeptides expressed in yeast hosts may be accomplished by theinclusion of nucleotide sequence encoding the yeast I-factor leadersequence at the 5′ end of the sel-10-encoding nucleotide sequence.

[0049] Insect host cell culture systems may also be used for theexpression of Sel-10 polypeptides. In a preferred embodiment, the sel-10polypeptides of the invention are expressed using a baculovirusexpression system. Further information regarding the use of baculovirussystems for the expression of heterologous proteins in insect cells arereviewed by Luckow and Summers, Bio/Technology 6:47 (1988).

[0050] In another preferred embodiment, the sel-10 polypeptide isexpressed in mammalian host cells. Non-limiting examples of suitablemammalian cell lines include the COS-7 line of monkey kidney cells(Gluzman et al., Cell 23:175 (1981)) and Chinese hamster ovary (CHO)cells.

[0051] The choice of a suitable expression vector for expression of thesel-10 polypeptides of the invention will of course depend upon thespecific mammalian host cell to be used, and is within the skill of theordinary artisan. Examples of suitable expression vectors include pcDNA3(Invitrogen) and pSVL (Pharmacia Biotech). Expression vectors for use inmammalian host cells may include transcriptional and translationalcontrol sequences derived from viral genomes. Commonly used promotersequences and enhancer sequences which may be used in the presentinvention include, but are not limited to, those derived from humancytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40(SV40). Methods for the construction of mammalian expression vectors aredisclosed, for example, in Okayama and Berg (Mol. Cell. Biol. 3:280(1983)); Cosman et al. (Mol. Immunol. 23:935 (1986)); Cosman et al.(Nature 312:768 (1984)); EP-A-0367566; and WO 91/18982.

[0052] The polypeptides of the present invention may also be used toraise polyclonal and monoclonal antibodies, which are useful indiagnostic assays for detecting sel-10 polypeptide expression. Suchantibodies may be prepared by conventional techniques. See, for example,Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, Kennetet al. (eds.), Plenum Press, New York (1980).

[0053] The sel-10 nucleic acid molecules of the present invention arealso valuable for chromosome identification, as they can hybridize witha specific location on a human chromosome. There is a current need foridentifying particular sites on the chromosome, as few chromosomemarking reagents based on actual sequence data (repeat polymorphisms)are presently available for marking chromosomal location. Once asequence has been mapped to a precise chromosomal location, the physicalposition of the sequence on the chromosome can be correlated withgenetic map data. The relationship between genes and diseases that havebeen mapped to the same chromosomal region can then be identifiedthrough linkage analysis, wherein the coinheritance of physicallyadjacent genes is determined. Whether a gene appearing to be related toa particular disease is in fact the cause of the disease can then bedetermined by comparing the nucleic acid sequence between affected andunaffected individuals.

[0054] The sel-10 polypeptides of the invention, and the DNA encodingthem, may also be used to further elucidate the biological mechanism ofAD, and may ultimately lead to the identification of compounds that canbe used to alter such mechanisms. The sel-10 polypeptides of theinvention are 47.6% identical and 56.7% similar to C. elegans sel-10. Asis described above, mutations to C. elegans sel-10 are known to suppressmutations to sel-12 that result in a loss-of-function for egg laying,and also to suppress certain hypomorphic mutations to lin-12. Mutationsto C. elegans sel-12 can also be rescued by either of the humanAD-linked genes PS-1 (42.7% identical to sel-12) or PS-2 (43.4%identical to sel-12). However, human PS-1 with a familial AD-linkedmutant has a reduced ability to rescue sel-12 mutants (Levitan, D. etal., Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)).

[0055] This demonstrated interchangeability of human and C. elegansgenes in the notch signaling pathway makes it reasonable to predict thatmutations of human sel-10 will suppress mutations to PS-1 or PS-2 thatlead to AD, especially in light of the predicted structure of sel-10. Asdescribed above, PS-1 and PS-2 mutations that lead to AD are those whichinterfere with the proteolytic processing of PS-1 or PS-2. The sel-10polypeptides of the invention are members of the β-transducin proteinfamily, which includes yeast CDC4, a component of an enzyme whichfunctions in the ubiquitin-dependent protein degradation pathway. Thus,human sel-10 may regulate presenilin degradation via theubiquitin-proteasome pathway. Alternatively, or in addition, humansel-10 may alter presenilin function by targeting for degradationthrough ubiquitination a modulator of presenilin activity, e.g., anegative regulator. Therefore, mutations to sel-10 may reverse thefaulty proteolytic processing of PS-1 or PS-2 which occurs as a resultof mutation to PS-1 or PS-2 or otherwise increase presenilin function.For the same reason, inhibition of sel-10 activity may also act toreverse PS-1 or PS-2 mutations. Thus, it may be hypothesized thatcompounds which inhibit either the expression or the activity of thehuman sel-10 polypeptides of the invention may reverse the effects ofmutations to PS-1 or PS-2, and thus be useful for the prevention ortreatment of AD.

[0056] Thus, C. elegans may be used as a genetic system for theidentification of agents capable of inhibiting the activity orexpression of the human sel-10 polypeptides of the invention. A suitableC. elegans strain for use in such assays lacks a gene encoding active C.elegans sel-10, and exhibits a loss-of-function for egg-laying resultingfrom an inactivated sel-12 gene. Construction of C. elegans strainshaving a loss-of-function for egg-laying due to mutation of sel-12 maybe accomplished using routine methods, as both the sequence of sel-12(Genebank accession number U35660) and mutations to sel-12 resulting ina loss-of-function for egg laying are known (see Levitan et al., Nature377: 351-354 (1995), which describes construction of C. eleganssel-12(ar171)). An example of how to make such a strain is also given inLevitan et al. (Nature 377: 351-354 (1995)). Wild-type C. elegans sel-10in the C. elegans sel-12(ar171)), is also mutagenized using routinemethods, such as the technique used for sel-12 mutagenesis in Levitan etal., supra.

[0057] In order to identify compounds inhibiting human sel-10 activity,a DNA vector containing a human sel-10 gene encoding any of thewild-type human sel-10 proteins of the invention is introduced into theabove-described C. elegans strain. In a preferred embodiment, theheterologous human sel-10 gene is integrated into the C. elegans genome.The gene is then expressed, using techniques described in Levitan et al.(Proc. Natl. Acad. Sci. USA 93: 14940-14944 (1996)). Test compounds arethen administered to this strain in order to determine whether a givenagent is capable of inhibiting sel-10 activity so as to suppressmutations to sel-12 or lin-12 that result in egg-laying defects.Egg-laying in this strain is then determined, e.g. by the assaydescribed in Levitan et al. (Proc. Natl. Acad. Sci. USA 93: 14940-14944(1996)). To confirm that the compound's effect on egg-laying is due toinhibition of sel-10 activity, the action of the compound can be testedin a second biochemical or genetic pathway that is known to be affectedby loss-of-function mutations in sel-10 (e.g., further elevation oflin-12 activity in lin-12(d) hypomorphic strains). Such assays may beperformed as described in Sundarem and Greenwald (Genetics 135: 765-783(1993)).

[0058] Alternatively, compounds are tested for their ability to inhibitthe E3 Ubiquitin Ligating Enzyme. Assays procedures for demonstratingsuch activity are well known, and involve reconstitution of theubiquitinating system using purified human sel-10 protein together withthe yeast proteins Cdc4p, Cdc53p and Skplp and an El enzyme, the E2enzyme Cdc34p, ubiquitin, a target protein and an ATP regeneratingsystem (Feldman et al., 1997). The sel-10 ubiquitination system may alsobe reconstituted with the C. elegans counterparts of the yeastcomponents, e.g., cul-1 (also known as lin-19) protein substituting forCdc53p (Kipreos et al., Cell 85:829 (1996)) and the protein F46A9substituting for Skplp, or with their mammalian counterparts, e.g.,Cul-2 protein substituting for Cdc53p (Kipreos et al., ibid.) andmammalian Skplp substituting for yeast Skp1p. A phosphorylation systemprovided by a protein kinase is also to be included in the assay systemas per Feldman et al., 1997.

[0059] Alternatively, cell lines which express human sel-10 due totransformation with a human sel-10 cDNA and which as a consequence haveelevated APP processing and formation of Aβ₁₋₄₀ or Aβ₁₋₄₂ may also beused for such assays as in Example 3. Compounds may be tested for theirability to reduce the elevated Aβ processing seen in the sel-10transformed cell line.

[0060] Compounds that rescue the egg-laying defect or that inhibit E3Ubiquitin Ligating Enzyme are then screened for their ability to cause areduction in the production of A-beta₁₋₄₀ or A-beta₁₋₄₂ in a human cellline. Test compounds are used to expose IMR-32 or other human cell linesknown to produce A-beta₁₋₄₀ or A-beta₁₋₄₂ (Asami-Okada et al.,Biochemistry 34: 10272-10278 (1995)), or in human cell lines engineeredto express human APP at high levels. In these assays, A-beta₁₋₄₀ orA-beta₁₋₄₂ is measured in cell extracts or after release into the mediumby ELISA or other assays which are known in the art (Borchelt et al.,Neuron 17: 1005-1013 (1996); Citron et al., Nat. Med. 3: 67-72 (1997)).

[0061] Having generally described the invention, the same will be morereadily understood by reference to the following examples, which areprovided by way of illustration and are not intended as limiting.

EXAMPLES Example 1 Identification of a Human Homologue to C. eleganssel-10

[0062] Results

[0063] Identification of sel-10 in ACEDB: Sel-10 maps between the clonedpolymorphisms arP3 and TCPARI just to the left of him-5 [ACEDB entrywm95p536]. Three phage lambda clones have been sequenced across theinterval, F53C11, F09F3, and F55B 12. Sel-10 is reported to havehomology to yeast cdc4 [ACEDB entry wm97ab259]. Blast search revealed asingle ORF with homology to yeast cdc4 (CC4_YST) within the intervaldefined by arP3 and TCPARI corresponding to the GenPep entry F55B12.3.F55B12.3, like yeast cdc4, is a member of the β-transducin proteinfamily. This family is characterized by the presence of multiple WD40repeats [Neer, E. J. et al., Nature 371: 297-300 (1994)].

[0064] Identification of a human sel-10 homologue, Incyte 028971: TheGenPep entry F55B12.3 was used to search the LifeSeq, LifeSeq FL andEMBL data bases using tblastn. The search revealed multiple homologiesto β-transducin family members including LIS-1 (S36113 and P43035), agene implicated in Miller-Dieker lissencephaly, a Xenopus laevis gene,TRCPXEN (U63921), and a human contig in LifeSeq FL, 028971. Since therealso are multiple β-transducin family members within the C. elegansgenome, these were collected using multiple blast searches and thenclustered with the sel-10 candidate genes. Multiple alignments wereperformed with the DNAStar program Megalign using the Clustal method.This revealed that LIS-1 clustered with T03F6.F, a differentβ-transducin family member and thus excluded it as a candidate sel-10homologue. TRCPXEN clustered with KIOB2.1, a gene which also clusterswith F55B12.3 and CC4YST, while Incyte 028971 clustered with sel-10.Thus, Incyte 028971 appears to encode the human homologue of C. eleganssel-10. Sequence homology betwcen sel-10 and 028Q71 is strongest in theregion of the protein containing 7 repeats of the WD40 motif. The Incyte028971 contig contains 44 ESTs from multiple libraries includingpancreas, lung, T-lymphocytes, fibroblasts, breast, hippocampus, cardiacmuscle, colon, and others.

[0065] Public EST: Blastx searches with the DNA sequence 028971 againstthe TREMBLP dataset identified a single homologous mouse EST (W85144)from the IMAGE Library, Soares mouse embryo NbME13.5-14.5. The blastxalignment of 028971 with W85144 and then with F55B12.3 revealed a changein reading frame in 028971 which probably is due to a sequencing error.

[0066] Blastn searches of the EMBL EST database with the 028971 DNAsequence revealed in addition to W85144, three human EST that align withthe coding sequence of 028971 and six EST that align with the 3′untranslated region of the 028971 sequence.

[0067] Protein Motifs: Two protein motifs were identified in F55B 12.3which are shared with yeast cdc4, mouse w85144 and human 028971. Theseare an F-box in the N-terminal domain and seven β-transducin repeats inthe C-terminal domain.

[0068] Discussion

[0069] The sel-10 gene encodes a member of the β-transducin proteinfamily. These are characterized by the presence of multiple WD40 repeats[Neer, E. J. et al., Nature 371: 297-300 (1994)]. The repeats are 20-40amino acids long and are bounded by gly-his (GH) and trp-asp (WD)residues. Solution of the three dimensional structure of β-transducinindicates that the WD40 repeats form the arms of a seven-bladedpropeller like structure [Sondek, J. et al., Nature 379: 369-74 (1996)].Each blade is formed by four alternating pleats of beta-sheet with apair of the conserved aspartic acid residues in the protein motifforming the limits of one internal beta strand. WD40 repeats are foundin over 27 different proteins which represent diverse functional classes[Neer, E. J. et al., Nature 371: 297-300 (1994)]. These regulatecellular functions including cell division, cell fate determination,gene transcription, signal transduction, protein degradation, mRNAmodification and vesicle fusion. This diversity in function has led tothe hypothesis that β-transducin family members provide a commonscaffolding upon which multiprotein complexes can be assembled.

[0070] The homology of sel-10, 28971 and W85144 to the yeast cdc4 genesuggests a functional role in the ubiquitin-proteasome pathway forintracellular degradation of protein. Mutations of the yeast cdc4 genecause cell cycle arrest by blocking degradation of Sicl, an inhibitor ofS-phase cyclin/cdk complexes [King, R. W. et al., Science 274: 1652-9(1996)]. Phosphorylation of Sic1 targets it for destruction through theubiquitin-proteasome pathway. This pathway consists of three linkedenzyme reactions that are catalyzed by multiprotein complexes[Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L.Schwartz, FASEB J. 8: 182-91 (1994)]. Initially, the C-terminal glycineof ubiquitin is activated by ATP to form a high energy thiol esterintermediate in a reaction catalyzed by the ubiquitin-activating enzyme,E1. Following activation, an E2 enzyme (ubiquitin conjugating enzyme)transfers ubiquitin from E1 to the protein target. In some cases, E2acts alone. In others, it acts in concert with an E3 ubiquitin-ligatingenzyme which binds the protein substrate and recruits an E2 to catalyzeubiquitination. E2 ubiquitin-conjugating enzymes comprise a fairlyconserved gene family, while E3 enzymes are divergent in sequence[Ciechanover, A., Cell 79: 13-21 (1994); Ciechanover, A. and A. L.Schwartz, FASEB J. 8: 182-91 (1994)].

[0071] In yeast, mutation of the E2 ubiquitin-conjugating enzyme, cdc34,causes cell cycle arrest through failure to degrade the Sic1 inhibitorof the S-phase cyclin/cdk complex [King, R. W. et al., Science 274:1652-9 (1996)]. Sicl normally is degraded as cells enter the G1-S phasetransition, but in the absence of cdc34, Sicl escapes degradation andits accumulation causes cell cycle arrest. Besides cdc34, cdc4 is one ofthree other proteins required for the G1-S phase transition. The othertwo are cdc53 and Skp1. As discussed above, cdc4 contains two structuralmotifs, seven WD40 repeats (which suggests that the protein forms abeta-propeller) and a structural motif shared with cyclin F which is aninteraction domain for Skp1 [Bai, C. et al., Cell 86: 263-74 (1996)].Insect cell lysates containing cdc53, cdc4 and skp1 (and also ubiquitin,cdc34 and E1) can transfer ubiquitin to Sic1 suggesting that one or moreof these components functions as an E3 ubiquitin-ligating enzyme [King,R. W. et al., Science 274: 1652-9 (1996)]. Increased expression ofeither cdc4 or Skp1 partially rescues loss of the other.

[0072] In C. elegans, mutation of sel-10 has no visible phenotypeindicating that sel-10 does not play a role in regulation of thecell-cycle. A closely related, C. elegans β-transducin family member,KIOB2.6 may play that role as it clusters with the gene TRCP_XEN fromXenopus laevis which rescues yeast cell cycle mutants arrested in lateanaphase due to a failure to degrade cyclin B [Spevak, W. et al., Mol.Cell. Biol. 13: 4953-66 (1993)]. If sel-10 does encode a component of anE3-ubiquitin ligating enzyme, how might it suppress sel-12 and enhancelin-12 mutations? The simplest hypothesis is that sel-10 regulatesdegradation of both proteins via the ubiquitin-proteasome pathway. Bothsel-12 and lin-12 are transmembrane proteins Sel-12 crosses the membrane8 times such that its N- and C-termini face the cytosol [Kim, T. W. etal., J. Biol. Chem. 272: 11006-10 (1997)], while lin-12 is a type 1transmembrane protein (Greenwald, I. and G. Seydoux, Nature 346: 197-9(1990)). Both are ubiquitinated, and in the case of human PS2, steadystate levels increase in cells treated with an inhibitor of theproteasome, N-acetyl-L-leucinal-L-norleucinal and lactacystin (Li, X.and I. Greenwald, Neuron. 17: 1015-21 (1996)). Alternatively, sel-10 maytarget for degradation of a negative regulator of presenilin function.

[0073] The genetic analysis and protein function suggested by homologyto cdc4 implies that drug inhibitors of human sel-10 may increase steadystate levels of human presenilins. This could potentiate activity of thepresenilin pathway and provide a means for therapeutic intervention inAlzheimer's disease.

Example 2 5′ RACE Cloning of a Human cDNA Encoding Sel-10, an ExtragenicSuppressor of Presenilin Mutations in C. elegans

[0074] Materials and Methods

[0075] Oligonucleotide primers for the amplification of the sel-10coding sequence from C. elegans cDNA were prepared based on the sequenceof F55B12.3, identified in Example 1 as the coding sequence for C.elegans sel-10. The primers prepared were:5′-CGGGATCCACCATGGATGATGGATCGATGACACC-3′ (SEQ ID NO:11) and5′-GGAATTCCTTAAGGGTATACAGCATCAAAGTCG-3′ (SEQ ID NO:12). C. elegans mRNAwas converted to cDNA using a BRL Superscript II Preamplification kit.The PCR product was digested with restriction enzymes BamHI and EcoRI(LTI, Gaithersberg, Md.) and cloned into pcDNA3.1 (Invitrogen). Twoisolates were sequenced (ABI, Perkin-Elmer Corp).

[0076] The sequence of Incyte clone 028971 (encoding a portion of thehuman homologue of C. elegans sel-10), was used to design four antisenseoligonucleotide primers: 5′-TCACTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:13),5′-GGTAA-TTACAAGTTCTTGTTGAACTG (SEQ ID NO: 14),5′-CCCTGCAACGTGTGT-AGACAGG-3′ (SEQ ID NO:15), and5′-CCAGTCTCTGCATTCCACACTTTG-3′ (SEQ ID NO:16) to amplify the missing 5′end of human sel-10. The Incyte LifeSeq “Electronic Northern” analysiswas used to identify tissues in which sel-10 was expressed. Two ofthese, hippocampus and mammary gland, were chosen for 5′ RACE cloningusing a CloneTech Marathon kit and prepared Marathon-ready cDNA fromhippocampus and mammary gland. PCR products were cloned into the TAvector pCR3.1 (Invitrogen), and isolates were sequenced. An alternate 5′oligonucleotide primer was also designed based on Incyte clones whichhave 5′ ends that differ from the hippocampal sel-10 sequence:5′-CTCAGACAGGTCAGGACATTTGG-3′ (SEQ ID NO:17).

[0077] Blastn was used to search Incyte databases LifeSeq and LifeSeqFL.Gap alignments and translations were performed with GCG programs(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, Madison Wis.).

[0078] Results

[0079] The coding sequence of the C. elegans sel-10: The predictedcoding sequence of the C. elegans sel-10, F55B12.3, had originally beendetermined at the Genome Sequencing Center, Washington University, St.Louis, by using the computer program GeneFinder to predict introns andexons in the genomic cosmid F55B 12. The hypothetical cDNA sequence wasconfirmed by amplifying this region from C. elegans cDNA, cloning, andsequencing it.

[0080] The coding sequence of the human sel-10 gene homologue: All ofthe 028971 antisense oligonucleotides amplified a 5′ RACE product fromhuman hippocampal and mammary cDNA. The longest PCR product from thehippocampal reactions was cloned and sequenced. This PCR reaction wasdesigned to generate products which end at the predicted stop codon. Twoisolates contained identical sequence which begins 880 bases before thebeginning of the 028971 sequence. This sequence was confirmed bycomparison with panning Incyte cDNA clones. The Incyte clones thatspanned the 5′ end of the human sel-10 homologue were not annotated asF55B12.3, as the homology in this region between the uman and C. elegansgenes is low, and as the overlap between these clones and the annotatedclones happened to be too small for them to be clustered in the Incytedatabase or uncovered by our blasting the Incyte database with the028971 sequence.

[0081] The predicted protein sequences of human sel-10 have 47.6%identity and 56.7% similarity to C. elegans sel-10. The N-terminus ofthe human sel-10 sequence begins with 4 in-frame methionines. Inaddition to the WD40 repeats described above, the human sequence alsocontains a region homologous to the CDC4 F-box for binding Skp1, asexpected for a sel-10 homologue.

[0082] Different Human Sel-10 mRNAs Expressed in Mammary and HippocampalTissues:

[0083] Several additional human sel-10 ESTs which differ from thehippocampal sequence were identified. These are an exact match, whichindicates that the alternative transcript is probably real. Comparisonof these sequences with the human hippocampal sel-10 sequence showsdivergence prior to the 4th in-frame methionine and then exact sequencematch thereafter. An oligonucleotide primer specific for the 5′ end ofthis alternative transcript was found to amplify a product from mammarybut not hippocampal cDNA. This indicates either that the human sel-10transcript undergoes differential splicing in a tissue-specific fashionor that the gene contains multiple, tissue specific promoters.

[0084] Discussion

[0085] 5′RACE and PCR amplification were used to clone a full-lengthcDNA encoding the human homologue of the C. elegans gene, sel-10.Sequence analysis confirms the earlier prediction that sel-10 is amember of the CDC4 family of proteins containing F-Box and WD40 Repeatdomains. Two variants of the human sel-10 cDNA were cloned fromhippocampus and mammary gland which differed in 5′ sequence precedingthe apparent site of translation initiation. This implies that the genemay have two or more start sites for transcription initiation which aretissue-specific or that the pattern of exon splicing is tissue-specific.

Example 3 Expression Of Epitope-Tagged Sel-10 In Human Cells, andPerturbation Of Amyloid β Peptide Processing By Human Sel-10

[0086] Materials And Methods

[0087] Construction of Epitope-Tagged Sel-10: Subcloning, Cell Growthand Transfection:

[0088] An EcoR1 site was introduced in-frame into the human sel-10 cDNAusing a polymerase chain reaction (PCR) primed with the oligonucleotides237 (5′-GGAATTC-CATGAAAAGATTGGACCATGGTTCTG-3′) (SEQ ID NO:18) and 206(5′-GGA-ATTCCTCACTTCATGT-CACATCAAAGTCCAG-3′) (SEQ ID NO:19). Theresulting PCR product was cloned into the EcoR1 site of the vectorpCS2+MT. This fused a 5′ 6-myc epitope tag in- frame to the fifthmethionine of the hippocampal sel-10 cDNA, i.e., upstream of nucleotide306 of the sequence given in SEQ ID NO:1. The nucleotide sequence ofthis construct, designated 6myc-N-sel-10, is given in SEQ ID NO: 20,while the amino acid sequence of the polypeptide encoded thereby isgiven in SEQ ID NO: 21. The hippocampal and mammary sel-10 cDNA divergeupstream of this methionine. A PS1 cDNA with a 3′-FLAG tag (PS1-C-FLAG)was subcloned into the pcDNA3.1 vector. An APP cDNA containing theSwedish NL mutation and an attenuated ER retention sequence consistingof the addition of a di-lysyl motif to the C-terminus of APP695(APP695NL-KK) was cloned into vector pIRES-EGFP (Mullan et al., NatGenet 1992 August; 1(5):345-7). HEK293 and IMR32 cells were grown to 80%confluence in DMEM with 10% FBS and transfected with the above cDNA. Atotal of 10 mg total DNA/6×10 cells was used for transfection with asingle plasmid. For cotransfections of multiple plasmids, an equalamount of each plasmid was used for a total of 10 mg DNA usingLipofectAmine (BRL).

[0089] In order to construct C-term V5 his tagged sel-10 and the C-termmychis tagged sel-10, the coding sequence of human hippocampal sel-10was amplified using oligonucleotides primers containing a KpnIrestriction site on the 5′ primer:5′-GGGTA-CCCCTCATTATTCCCTCGAGTTCTTC-3′ (SEQ ID NO:22) and an EcoRI siteon the 3′ primer: 5′-GGAATTCCTTCATGTCCACATCAAAGTCC-3′ (SEQ ID NO:23),using the original human sel-10 RACE pcr product as template. Theproduct was digested with both KpnI and EcoRI and cloned into either thevector pcDNA6/V5-His A or pcDNA3.1/Myc-His(+) A (Invitrogen). Thenucleotide sequence of independent isolates was confirmed by dideoxysequencing. The nucleotide sequence of the C-term V5 his tagged sel-10is given in SEQ ID NO: 24, while the amino acid sequence of thepolypeptide encoded thereby is given in SEQ ID NO: 25. The nucleotidesequence of independent isolates was confirmed by dideoxy sequencing.The nucleotide sequence of the C-term mychis tagged sel-10 is given inSEQ ID NO: 26, while the amino acid sequence of the polypeptide encodedthereby is given in SEQ ID NO: 27.

[0090] Clonal Selection of transformed cells by FACS: Cell samples wereanalyzed on an EPICS Elite ESP flow cytometer (Coulter, Hialeah, FL)equipped with a 488 nm excitation line supplied by an air-cooled argonlaser. EGFP emission was measured through a 525 nm band-pass filter andfluorescence intensity was displayed on a 4-decade log scale aftergating on viable cells as determined by forward and right angle lightscatter. Single green cells were separated into each well of one 96 wellplate containing growth medium without G418. After a four day recoveryperiod, G418 was added to the medium to a final concentration of 400mg/ml. Wells with clones were expanded from the 96 well plate to a 24well plate and then to a 6 well plate with the fastest growing colonieschosen for expansion at each passage.

[0091] Immunofluorescence: Cells grown on slides were fixed 48 hrs aftertransfection with 4% formaldehyde and 0.1% Triton X-100 in PBS for 30min on ice and blocked with 10% Goat serum in PBS (blocking solution) 1hr RT (i.e., 25° C.), followed by incubation with mouse anti-myc (10mg/ml) or rabbit anti-FLAG (0.5 mg/ml) antibody 4° C. O/N and thenfluorescein-labeled goat anti-mouse or anti-rabbit antibody (5 mg/ml) inblocking solution 1 hr at 25° C.

[0092] Western blotting: Cell lysates were made 48 hrs aftertransfection by incubating 10⁵ cells with 100 ml TENT (50 mM Tris-HCl pH8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, 1× protease inhibitorcocktail) 10 min on ice followed by centrifugation at 14,000 g. Thesupernatant was loaded on 4-12% NuPage gels (50 mg protein/lane) andelectrophoresis and transfer were conducted using an Xcell II Mini-Cellsystem (Novex). The blot was blocked with 5% milk in PBS 1 hr RT andincubated with anti-myc or anti-FLAG antibody (described in“Immunofluorescence” above) 4° C. O/N, then sheep anti-mouse oranti-rabbit antibody-HRP (0.1 mg/ml) 1 hr RT, followed by Supersignal(Pierce) detection.

[0093] ELISA: Cell culture supernatant or cell lysates (100 ml formicacid/10⁶ cells) were assayed in the following double antibody sandwichELISA, which is capable of detecting levels of Aβ₁₋₄₀ and Aβ₁₋₄₂ peptidein culture supernatant.

[0094] Human Aβ 1-40 or 1-42 was measured using monoclonal antibody(mAb) 6E10 (Senetek, St. Louis, Mo.) and biotinylated rabbit antiserum162 or 164 (NYS Institute for Basic Research, Staten Island, N.Y.) in adouble antibody sandwich ELISA. The capture antibody 6E10 is specific toan epitope present on the N-terminal amino acid residues 1-16 of hAβ.The conjugated detecting antibodies 162 and 164 are specific for hAβ1-40 and 1-42, respectively. The sandwich ELISA was performed accordingto the method of Pirttila et al. (Neurobiology of Aging 18: 121-7(1997)). Briefly, a Nunc Maxisorp 96 well immunoplate was coated with100 μl/well of mAb 6E 10 (5 μg/ml) diluted in 0.1 Mcarbonate-bicarbonate buffer, pH 9.6 and incubated at 4° C. overnight.After washing the plate 3× with 0.01M DPBS (Modified Dulbecco'sPhosphate Buffered Saline (0.008M sodium phosphate, 0.002M potassiumphosphate, 0.14M sodium chloride, 0.01 M potassium chloride, pH 7.4)from Pierce, Rockford, Ill.) containing 0.05% of Tween-20 (DPBST), theplate was blocked for 60 min with 200 μl of 10% normal sheep serum(Sigma) in 0.01M DPBS to avoid non-specific binding. Human Aβ 1-40 or1-42 standards 100 μl/well (Bachem, Torrance, Calif.) diluted, from a 1mg/ml stock solution in DMSO, in non transfected conditioned cell mediumwas added after washing the plate, as well as 100 μl/well of sample i.e.filtered conditioned medium of transfected cells. The plate wasincubated for 2 hours at room temperature and 4° C. overnight. The nextday, after washing the plate, 100 μl/well biotinylated rabbit antiserum162 1:400 or 164 1:50 diluted in DPBST +0.5% BSA was added and incubatedat room temperature for 1 hr 15 min. Following washes, 100 μl/wellneutravidin-horseradish peroxidase (Pierce, Rockford, 11) diluted1:10,000 in DPBST was applied and incubated for 1 hr at roomtemperature. After the last washes 100 μl/well of o-phenylnediaminedihydrochloride (Sigma Chemicals, St. Louis, Mo.) in 50 mM citricacid/100 mM sodium phosphate buffer (Sigma Chemicals, St. Louis, Mo.),pH 5.0, was added as substrate and the color development was monitoredat 450 nm in a kinetic microplate reader for 20 min. using Soft max Prosoftware.

[0095] Results

[0096] Transfection of HEK293 cells: Transfection efficiency wasmonitored through the use of vectors that express green fluorescentprotein (GFP) or by immunofluorescent detection of epitope-tagged sel-10or PS1. An N-terminal 6-myc epitope was used to tag human sel-10(6myc-N-sel-10), while PS1 was tagged with a C-terminal FLAG epitope(PS1-C-FLAG). APP695 was modified by inclusion of the Swedish NLmutation to increase Aβ processing and an attenuated endoplasmicreticulum (ER) retention signal consisting of a C-terminal di-lysinemotif (APP695NL-KK). The di-lysine motif increases Aβ processing abouttwo fold. The APP695NL-KK construct was inserted into the first cistronof a bicistronic vector containing GFP (pIRES-EGFP, Invitrogen) to allowus to monitor transfection efficiency. Transfection efficiency in HEK293cells was about 50% for transfections with a single plasmid DNA. Forcotransfections with two plasmids, about 30-40% of the cells expressedboth proteins as detected by double immunofluorescence.

[0097] Expression of recombinant protein in transfected HEK293 cells wasconfirmed by Western blot as illustrated for PS 1-C-FLAG and6myc-N-sel-10 (FIG. 1A). In the case of cotransfections with threeplasmids (PS1-C-FLAG+6myc-N-sel-0+APP), all three proteins were detectedin the same cell lysate by Western blot (FIG. 1B) using appropriateantibodies.

[0098] Effect of 6myc-N-sel-10 and PS1-C-FLAG on Aβ processing:Cotransfection of APP695NL-KK with 6myc-N-sel-10 or PS1-C-FLAG intoHEK293 cells increased the release of Ab1-40 and Ab1-42 peptide into theculture supernatant by 2- to 3-fold over transfections with justAPP695NL-KK (Table 1). Cotransfection of APP695NL-KK with both6myc-N-sel-10 and PS1-C-FLAG increased Ab release still further (i.e.,4- to 6-fold increase). In contrast, the ratio of Ab 1-42/(Ab 1-40+Ab1-42) released into the supernatant decreased about 50%. The subtledecrease in the ratio of Ab1-42/(Ab1-40+Ab1-42) reflects the largerincrease in Ab 1-40 relative to Ab 1-42. Neither 6myc-N-sel-10 norPS1-C-FLAG affected endogenous Ab production in HEK293 cells. Similarobservations were also obtained in IMR32 cells (Table 2). However, IMR32cells transfected less well than HEK293 cells, so the stimulation ofAPP695NL-KK processing by cotransfection with 6myc-N-sel-10 orPS1-C-FLAG was lower.

[0099] Levels of Ab 1-40 expressed in HEK293 cells transfected withAPP695NL-KK were sufficient to measure Ab peptide in both the culturesupernatant and cell pellet. Considerably more Ab 1-40 is detected inthe HEK293 cell pellet than in the supernatant in cells transfected withjust APP695NL-KK. Cotransfection with 6myc-N-sel-10 or PS1-C-FLAGproportionately decreased Ab 1-40 in the cell pellet and increased Ab inthe culture supernatant. This implies that 6myc-N-sel-10 and PS1-C-FLAGalter processing or trafficking of APP such that proportionately more Abis released from the cell.

[0100] Effect of 6myc-N-sel-10 and PS1-C-FLAG expression on endogenousAβ processing: The effect of 6myc-N-sel-10 on the processing ofendogenous APP by human cells was assessed by creating stablytransformed HEK293 cell lines expressing these proteins. Two cell linesexpressing 6myc-N-sel-10 were derived (sel-10/2 & sel-10/6) as well as acontrol cell line transformed with pcDNA3.1 vector DNA. Both6myc-N-sel-10 cell lines expressed the protein as shown by Western blotanalysis. Endogenous production of Ab 1-40 was increased in both6myc-N-sel-10 cell lines in contrast to vector DNA transformed cellsTable 3). In addition, stable expression of 6myc-N-sel-10 significantlyincreased Ab production after transfection with APP695NL-KK plasmid DNA(Table 3). Similar results were obtained with 6 stable cell linesexpressing PS 1-C-FLAG. All 6 cell lines showed significant elevation ofendogenous Aβ processing and all also showed enhanced processing of Abafter transfection with APP695NL-KK (Table 3). In addition, the increaseof Aβ processing seen with 6myc-N-sel-10 was also seen with sel-10tagged at the C-terminus with either mychis or v5his (See Table 4). BothC-terminal and N-terminal tags resulted in an increase in Aβ processing.

[0101] Discussion

[0102] These data suggest that, when over expressed, 6myc-N-sel-10 aswell as PS 1-C-FLAG alter Aβ processing in both transient and stableexpression systems. A 6-myc epitope tag was used in these experiments toallow detection of sel-10 protein expression by Western blot analysis.If as its sequence homology to yeast CDC4 suggests, sel-10 is an E2-E3ubiquitin ligase, it should be possible to identify the proteins ittargets for ubiquitination. Since the presenilins are degraded via theubiquitin-proteasome pathway, PS1 & PS2 are logical targets of sel-10catalyzed ubiquitination [Kim et al., J. Biol. Chem. 272:11006-11010(1997)]. How sel-10 affects Aβ processing is not understood at thispoint. In the future, it will be necessary to determine if sel-10 & PS1increase Aβ processing by altering production, processing, transport, orturn-over of APP, and whether the effect of PS1 is mediated or regulatedby sel-10.

[0103] These experiments suggest that sel-10 is a potential drug targetfor decreasing Ab levels in the treatment of AD. They also show that C.elegans is an excellent model system in which to investigate presenilinbiology in the context of AD. Thus, as is shown by cotransfectionexperiments, as well as in stable transformants, expression of6myc-N-sel 10 or PS 1-C-FLAG increases Aβ processing. An increase in Aβprocessing was seen in both HEK293 cells and IMR32 cells aftercotransfection of 6myc-N-sel10 or PS1-C-FLAG with APP695NL-KK. Instabletransformants of HEK293 cells expressing 6myc-Sel10 or PS1-C-FLAG, anincrease in endogenous Aβ processing was observed, as well as anincrease in Aβ processing after transfection with APP695NL-KK. Thissuggests that inhibitors of either sel-10 and/or PS1, may decrease Aβprocessing, and could have therappeutic potential for Alzheimer'sdisease.

[0104] It will be clear that the invention may be practiced otherwisethan as particularly described in the foregoing description andexamples.

[0105] Numerous modifications and variations of the present inventionare possible in light of the above teachings and, therefore, are withinthe scope of the invention.

[0106] The entire disclosure of all publications cited herein are herebyincorporated by reference. TABLE 1 Effect of 6myc-N-sel-10 andPS1-C-FLAG transient transfection on Ab levels in HEK293 cellsupernatants. Plasmids Ab1-42/total Ab Transfected Ab1-42 ng/ml Ab1-40ng/ml ng/ml pcDNA3  81 ± 20  231 ± 50 0.26 ± 0.05 6myc-N-sel-10  67 ± 7 246 ± 34 0.21 ± 0.03 PS1-C-FLAG  75 ± 18  227 ± 45 0.25 ± 0.03PS1-C-FLAG +  77 ± 21  220 ± 26 0.25 ± 0.03 6myc-N-sel-10 APP695NL-KK141 ± 27  896 ± 103 0.14 ± 0.02 APP695NL-KK + 308 ± 17 2576 ± 190 0.11 ±0.00 6myc-N-sel-10 APP695NL-KK + 364 ± 39 3334 ± 337 0.09 ± 0.00PS1-C-FLAG APP695NL-KK + 550 ± 20 5897 ± 388 0.09 ± 0.00 PS1-C-FLAG +6myc-N-sel-10

[0107] TABLE 2 Effect of 6myc-N-sel-10 and PS1-C-FLAG transienttransfection on Ab levels in IMR32 cell supernatants. PlasmidsAb1-42/total Ab Transfected Ab1-42 ng/ml Ab1-40 ng/ml ng/ml pcDNA3 65 ±3 319 ± 146 0.19 ± 0.06 6myc-N-sel-10 63 ± 0 246 ± 53 0.21 ± 0.04PS1-C-FLAG 67 ± 6 307 ± 79 0.18 ± 0.04 PS1-C-FLAG + 67 ± 6 302 ± 94 0.20± 0.08 6myc-N-sel-10 APP695NL-KK 66 ± 5 348 ± 110 0.17 ± 0.05APP695NL-KK + 75 ± 18 448 ± 141 0.15 ± 0.03 6myc-N-sel-10 APP695NL-KK +63 ± 26 466 ± 72 0.12 ± 0.02 PS1-C-FLAG APP695NL-KK + 81 ± 26 565 ± 1790.12 ± 0.01 PS1-C-FLAG + 6myc-N-sel-10

[0108] TABLE 3 Endogenous and exogenous Ab1-40 and Ab1-42 levels insupernatants from stable transformants of HEK293 cells. GFP APP695NL-KKTransfection Transfection Ab1-40 Ab1-42 Ab1-40 Ab1-42 Stable Line ng/mlng/ml ng/ml ng/ml 6myc-N-sel10/2 297 ± 29 109 ± 17 4877 ± 547  750 ± 326myc-N-sel10/6 168 ± 18  85 ± 11 8310 ± 308 1391 ± 19 PS1-C-FLAG/2  97 ±6  68 ± 8 3348 ± 68  493 ± 21 PS1-C-FLAG/8 118 ± 11  85 ± 17 3516 ± 364 515 ± 36 PS1-C-FLAG/9  83 ± 20  67 ± 16 2369 ± 73  350 ± 12PS1-C-FLAG/11 152 ± 17  68 ± 13 4771 ± 325  599 ± 25 PS1-C-FLAG/12 141 ±12  50 ± 10 4095 ± 210  449 ± 21 PS1-C-FLAG/13 270 ± 139  61 ± 28 6983 ±304  745 ± 41 pcDNA3/1  43 ± 13  75 ± 15 1960 ± 234  61 ± 6

[0109] TABLE 4 Sel-10 constructs with epitope tags at the N or Cterminus increase Aβ 1-40 and Aβ 1-42. construct Aβ 1-40 % increaseP-value Aβ 1-42 % increase P-value pcDNA 4240 ± 102 614 ± 106myc-N-sel-10 7631 ± 465 80% 3.7 × 10⁻⁶ 1136 ± 73  46% 7.9 × 10⁻⁶sel-10-C-mychis 5485 ± 329 29% 1.8 × 10⁻⁴ 795 ± 50 29% 4.0 × 10⁻⁴sel-10-C-V5his 6210 ± 498 46% 1.2 × 10⁻⁴ 906 ± 73 48% 2.1 × 10⁻⁴

[0110]

1 27 1 3550 DNA Homo sapiens unsure (2485) unsure (3372) 1 ctcattattccctcgagttc ttctcagtca agctgcatgt atgtatgtgt gtcccgagaa 60 gcggtttgatactgagctgc atttgccttt actgtggagt tttgttgccg gttctgctcc 120 ctaatcttccttttctgacg tgcctgagca tgtccacatt agaatctgtg acatacctac 180 ctgaaaaaggtttatattgt cagagactgc caagcagccg gacacacggg ggcacagaat 240 cactgaaggggaaaaataca gaaaatatgg gtttctacgg cacattaaaa atgatttttt 300 acaaaatgaaaagaaagttg gaccatggtt ctgaggtccg ctctttttct ttgggaaaga 360 aaccatgcaaagtctcagaa tatacaagta ccactgggct tgtaccatgt tcagcaacac 420 caacaacttttggggacctc agagcagcca atggccaagg gcaacaacga cgccgaatta 480 catctgtccagccacctaca ggcctccagg aatggctaaa aatgtttcag agctggagtg 540 gaccagagaaattgcttgct ttagatgaac tcattgatag ttgtgaacca acacaagtaa 600 aacatatgatgcaagtgata gaaccccagt ttcaacgaga cttcatttca ttgctcccta 660 aagagttggcactctatgtg ctttcattcc tggaacccaa agacctgcta caagcagctc 720 agacatgtcgctactggaga attttggctg aagacaacct tctctggaga gagaaatgca 780 aagaagaggggattgatgaa ccattgcaca tcaagagaag aaaagtaata aaaccaggtt 840 tcatacacagtccatggaaa agtgcataca tcagacagca cagaattgat actaactgga 900 ggcgaggagaactcaaatct cctaaggtgc tgaaaggaca tgatgatcat gtgatcacat 960 gcttacagttttgtggtaac cgaatagtta gtggttctga tgacaacact ttaaaagttt 1020 ggtcagcagtcacaggcaaa tgtctgagaa cattagtggg acatacaggt ggagtatggt 1080 catcacaaatgagagacaac atcatcatta gtggatctac agatcggaca ctcaaagtgt 1140 ggaatgcagagactggagaa tgtatacaca ccttatatgg gcatacttcc actgtgcgtt 1200 gtatgcatcttcatgaaaaa agagttgtta gcggttctcg agatgccact cttagggttt 1260 gggatattgagacaggccag tgtttacatg ttttgatggg tcatgttgca gcagtccgct 1320 gtgttcaatatgatggcagg agggttgtta gtggagcata tgattttatg gtaaaggtgt 1380 gggatccagagactgaaacc tgtctacaca cgttgcaggg gcatactaat agagtctatt 1440 cattacagtttgatggtatc catgtggtga gtggatctct tgatacatca atccgtgttt 1500 gggatgtggagacagggaat tgcattcaca cgttaacagg gcaccagtcg ttaacaagtg 1560 gaatggaactcaaagacaat attcttgtct ctgggaatgc agattctaca gttaaaatct 1620 gggatatcaaaacaggacag tgtttacaaa cattgcaagg tcccaacaag catcagagtg 1680 ctgtgacctgtttacagttc aacaagaact ttgtaattac cagctcagat gatggaactg 1740 taaaactatgggacttgaaa acgggtgaat ttattcgaaa cctagtcaca ttggagagtg 1800 gggggagtgggggagttgtg tggcggatca gagcctcaaa cacaaagctg gtgtgtgcag 1860 ttgggagtcggaatgggact gaagaaacca agctgctggt gctggacttt gatgtggaca 1920 tgaagtgaagagcagaaaag atgaatttgt ccaattgtgt agacgatata ctccctgccc 1980 ttccccctgcaaaaagaaaa aaagaaaaga aaaagaaaaa aatcccttgt tctcagtggt 2040 gcaggatgttggcttggggc aacagattga aaagacctac agactaagaa ggaaaagaag 2100 aagagatgacaaaccataac tgacaagaga ggcgtctgct gtctcatcac ataaaaggct 2160 tcacttttgactgagggcag ctttgcaaaa tgagactttc taaatcaaac caggtgcaat 2220 tatttctttattttcttctc cagtggtcat tggggcagtg ttaatgctga aacatcatta 2280 cagattctgctagcctgttc ttttaccact gacagctaga cacctagaaa ggaactgcaa 2340 taatatcaaaacaagtactg gttgactttc taattagaga gcatctgcaa caaaaagtca 2400 tttttctggagtggaaaagc ttaaaaaaat tactgtgaat tgtttttgta cagttatcat 2460 gaaaagcttttttttttatt ttttngccaa ccattgccaa tgtcaatcaa tcacagtatt 2520 agcctctgttaatctattta ctgttgcttc catatacatt cttcaatgca tatgttgctc 2580 aaaggtggcaagttgtcctg ggttctgtga gtcctgagat ggatttaatt cttgatgctg 2640 gtgctagaagtaggtcttca aatatgggat tgttgtccca accctgtact gtactcccag 2700 tggccaaacttatttatgct gctaaatgaa agaaagaaaa aagcaaatta ttttttttat 2760 tttttttctgctgtgacgtt ttagtcccag actgaattcc aaatttgctc tagtttggtt 2820 atggaaaaaagactttttgc cactgaaact tgagccatct gtgcctctaa gaggctgaga 2880 atggaagagtttcagataat aaagagtgaa gtttgcctgc aagtaaagaa ttgagagtgt 2940 gtgcaaagcttattttcttt tatctgggca aaaattaaaa cacattcctt ggaacagagc 3000 tattacttgcctgttctgtg gagaaacttt tctttttgag ggctgtggtg aatggatgaa 3060 cgtacatcgtaaaactgaca aaatatttta aaaatatata aaacacaaaa ttaaaataaa 3120 gttgctggtcagtcttagtg ttttacagta tttgggaaaa caactgttac agttttattg 3180 ctctgagtaactgacaaagc agaaactatt cagtttttgt agtaaaggcg tcacatgcaa 3240 acaaacaaaatgaatgaaac agtcaaatgg tttgcctcat tctccaagag ccacaactca 3300 agctgaactgtgaaagtggt ttaacactgt atcctaggcg atcttttttc ctccttctgt 3360 ttatttttttgnttgtttta tttatagtct gatttaaaac aatcagattc aagttggtta 3420 attttagttatgtaacaacc tgacatgatg gaggaaaaca acctttaaag ggattgtgtc 3480 tatggtttgattcacttaga aattttattt tcttataact taagtgcaat aaaatgtgtt 3540 ttttcatgtt3550 2 3571 DNA Homo sapiens unsure (2506) unsure (3393) 2 ctcagcaggtcaggacattt ggtaggggaa ggttgaaaga caaaagcagc aggccttggg 60 ttctcagccttttaaaaact attattaaat atatattttt aaaatttagt ggttagagct 120 tttagtaatgtgcctgtatt acatgtagag agtattcgtc aaccaagagg agttttaaaa 180 tgtcaaaaccgggaaaacct actctaaacc atggcttggt tcctgttgat cttaaaagtg 240 caaaagagcctctaccacat caaaccgtga tgaagatatt tagcattagc atcattgccc 300 aaggcctccctttttgtcga agacggatga aaagaaagtt ggaccatggt tctgaggtcc 360 gctctttttctttgggaaag aaaccatgca aagtctcaga atatacaagt accactgggc 420 ttgtaccatgttcagcaaca ccaacaactt ttggggacct cagagcagcc aatggccaag 480 ggcaacaacgacgccgaatt acatctgtcc agccacctac aggcctccag gaatggctaa 540 aaatgtttcagagctggagt ggaccagaga aattgcttgc tttagatgaa ctcattgata 600 gttgtgaaccaacacaagta aaacatatga tgcaagtgat agaaccccag tttcaacgag 660 acttcatttcattgctccct aaagagttgg cactctatgt gctttcattc ctggaaccca 720 aagacctgctacaagcagct cagacatgtc gctactggag aattttggct gaagacaacc 780 ttctctggagagagaaatgc aaagaagagg ggattgatga accattgcac atcaagagaa 840 gaaaagtaataaaaccaggt ttcatacaca gtccatggaa aagtgcatac atcagacagc 900 acagaattgatactaactgg aggcgaggag aactcaaatc tcctaaggtg ctgaaaggac 960 atgatgatcatgtgatcaca tgcttacagt tttgtggtaa ccgaatagtt agtggttctg 1020 atgacaacactttaaaagtt tggtcagcag tcacaggcaa atgtctgaga acattagtgg 1080 gacatacaggtggagtatgg tcatcacaaa tgagagacaa catcatcatt agtggatcta 1140 cagatcggacactcaaagtg tggaatgcag agactggaga atgtatacac accttatatg 1200 ggcatacttccactgtgcgt tgtatgcatc ttcatgaaaa aagagttgtt agcggttctc 1260 gagatgccactcttagggtt tgggatattg agacaggcca gtgtttacat gttttgatgg 1320 gtcatgttgcagcagtccgc tgtgttcaat atgatggcag gagggttgtt agtggagcat 1380 atgattttatggtaaaggtg tgggatccag agactgaaac ctgtctacac acgttgcagg 1440 ggcatactaatagagtctat tcattacagt ttgatggtat ccatgtggtg agtggatctc 1500 ttgatacatcaatccgtgtt tgggatgtgg agacagggaa ttgcattcac acgttaacag 1560 ggcaccagtcgttaacaagt ggaatggaac tcaaagacaa tattcttgtc tctgggaatg 1620 cagattctacagttaaaatc tgggatatca aaacaggaca gtgtttacaa acattgcaag 1680 gtcccaacaagcatcagagt gctgtgacct gtttacagtt caacaagaac tttgtaatta 1740 ccagctcagatgatggaact gtaaaactat gggacttgaa aacgggtgaa tttattcgaa 1800 acctagtcacattggagagt ggggggagtg ggggagttgt gtggcggatc agagcctcaa 1860 acacaaagctggtgtgtgca gttgggagtc ggaatgggac tgaagaaacc aagctgctgg 1920 tgctggactttgatgtggac atgaagtgaa gagcagaaaa gatgaatttg tccaattgtg 1980 tagacgatatactccctgcc cttccccctg caaaaagaaa aaaagaaaag aaaaagaaaa 2040 aaatcccttgttctcagtgg tgcaggatgt tggcttgggg caacagattg aaaagaccta 2100 cagactaagaaggaaaagaa gaagagatga caaaccataa ctgacaagag aggcgtctgc 2160 tgtctcatcacataaaaggc ttcacttttg actgagggca gctttgcaaa atgagacttt 2220 ctaaatcaaaccaggtgcaa ttatttcttt attttcttct ccagtggtca ttggggcagt 2280 gttaatgctgaaacatcatt acagattctg ctagcctgtt cttttaccac tgacagctag 2340 acacctagaaaggaactgca ataatatcaa aacaagtact ggttgacttt ctaattagag 2400 agcatctgcaacaaaaagtc atttttctgg agtggaaaag cttaaaaaaa ttactgtgaa 2460 ttgtttttgtacagttatca tgaaaagctt ttttttttat tttttngcca accattgcca 2520 atgtcaatcaatcacagtat tagcctctgt taatctattt actgttgctt ccatatacat 2580 tcttcaatgcatatgttgct caaaggtggc aagttgtcct gggttctgtg agtcctgaga 2640 tggatttaattcttgatgct ggtgctagaa gtaggtcttc aaatatggga ttgttgtccc 2700 aaccctgtactgtactccca gtggccaaac ttatttatgc tgctaaatga aagaaagaaa 2760 aaagcaaattatttttttta ttttttttct gctgtgacgt tttagtccca gactgaattc 2820 caaatttgctctagtttggt tatggaaaaa agactttttg ccactgaaac ttgagccatc 2880 tgtgcctctaagaggctgag aatggaagag tttcagataa taaagagtga agtttgcctg 2940 caagtaaagaattgagagtg tgtgcaaagc ttattttctt ttatctgggc aaaaattaaa 3000 acacattccttggaacagag ctattacttg cctgttctgt ggagaaactt ttctttttga 3060 gggctgtggtgaatggatga acgtacatcg taaaactgac aaaatatttt aaaaatatat 3120 aaaacacaaaattaaaataa agttgctggt cagtcttagt gttttacagt atttgggaaa 3180 acaactgttacagttttatt gctctgagta actgacaaag cagaaactat tcagtttttg 3240 tagtaaaggcgtcacatgca aacaaacaaa atgaatgaaa cagtcaaatg gtttgcctca 3300 ttctccaagagccacaactc aagctgaact gtgaaagtgg tttaacactg tatcctaggc 3360 gatcttttttcctccttctg tttatttttt tgnttgtttt atttatagtc tgatttaaaa 3420 caatcagattcaagttggtt aattttagtt atgtaacaac ctgacatgat ggaggaaaac 3480 aacctttaaagggattgtgt ctatggtttg attcacttag aaattttatt ttcttataac 3540 ttaagtgcaataaaatgtgt tttttcatgt t 3571 3 627 PRT Homo sapiens 3 Met Cys Val ProArg Ser Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly ValLeu Leu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu SerMet Ser Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu TyrCys Gln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser LeuLys Gly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80 Leu LysMet Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85 90 95 Glu ValArg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 100 105 110 TyrThr Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135140 Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145150 155 160 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp GluLeu 165 170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met GlnVal Ile 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu ProLys Glu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys AspLeu Leu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu AlaGlu Asp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu GlyIle Asp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys ProGly Phe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln HisArg Ile Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro LysVal Leu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln PheCys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn Thr LeuLys Val Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330 335 Leu ValGly His Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn 340 345 350 IleIle Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 355 360 365Glu Thr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375380 Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385390 395 400 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu HisVal 405 410 415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr AspGly Arg 420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys ValTrp Asp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly HisThr Asn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val ValSer Gly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val GluThr Gly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu ThrSer Gly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn AlaAsp Ser Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys LeuGln Thr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr CysLeu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp AspGly Thr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575 Ile ArgAsn Leu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580 585 590 TrpArg Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 595 600 605Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 610 615620 Asp Met Lys 625 4 592 PRT Homo sapiens 4 Met Ser Thr Leu Glu Ser ValThr Tyr Leu Pro Glu Lys Gly Leu Tyr 1 5 10 15 Cys Gln Arg Leu Pro SerSer Arg Thr His Gly Gly Thr Glu Ser Leu 20 25 30 Lys Gly Lys Asn Thr GluAsn Met Gly Phe Tyr Gly Thr Leu Lys Met 35 40 45 Ile Phe Tyr Lys Met LysArg Lys Leu Asp His Gly Ser Glu Val Arg 50 55 60 Ser Phe Ser Leu Gly LysLys Pro Cys Lys Val Ser Glu Tyr Thr Ser 65 70 75 80 Thr Thr Gly Leu ValPro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp 85 90 95 Leu Arg Ala Ala AsnGly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser 100 105 110 Val Gln Pro ProThr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser 115 120 125 Trp Ser GlyPro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser 130 135 140 Cys GluPro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln 145 150 155 160Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr 165 170175 Val Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr 180185 190 Cys Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu195 200 205 Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys ArgArg 210 215 220 Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys SerAla Tyr 225 230 235 240 Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg ArgGly Glu Leu Lys 245 250 255 Ser Pro Lys Val Leu Lys Gly His Asp Asp HisVal Ile Thr Cys Leu 260 265 270 Gln Phe Cys Gly Asn Arg Ile Val Ser GlySer Asp Asp Asn Thr Leu 275 280 285 Lys Val Trp Ser Ala Val Thr Gly LysCys Leu Arg Thr Leu Val Gly 290 295 300 His Thr Gly Gly Val Trp Ser SerGln Met Arg Asp Asn Ile Ile Ile 305 310 315 320 Ser Gly Ser Thr Asp ArgThr Leu Lys Val Trp Asn Ala Glu Thr Gly 325 330 335 Glu Cys Ile His ThrLeu Tyr Gly His Thr Ser Thr Val Arg Cys Met 340 345 350 His Leu His GluLys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu 355 360 365 Arg Val TrpAsp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly 370 375 380 His ValAla Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val 385 390 395 400Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu 405 410415 Thr Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu 420425 430 Gln Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile435 440 445 Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu ThrGly 450 455 460 His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn IleLeu Val 465 470 475 480 Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp AspIle Lys Thr Gly 485 490 495 Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn LysHis Gln Ser Ala Val 500 505 510 Thr Cys Leu Gln Phe Asn Lys Asn Phe ValIle Thr Ser Ser Asp Asp 515 520 525 Gly Thr Val Lys Leu Trp Asp Leu LysThr Gly Glu Phe Ile Arg Asn 530 535 540 Leu Val Thr Leu Glu Ser Gly GlySer Gly Gly Val Val Trp Arg Ile 545 550 555 560 Arg Ala Ser Asn Thr LysLeu Val Cys Ala Val Gly Ser Arg Asn Gly 565 570 575 Thr Glu Glu Thr LysLeu Leu Val Leu Asp Phe Asp Val Asp Met Lys 580 585 590 5 553 PRT Homosapiens 5 Met Gly Phe Tyr Gly Thr Leu Lys Met Ile Phe Tyr Lys Met LysArg 1 5 10 15 Lys Leu Asp His Gly Ser Glu Val Arg Ser Phe Ser Leu GlyLys Lys 20 25 30 Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr Thr Gly Leu ValPro Cys 35 40 45 Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala Ala AsnGly Gln 50 55 60 Gly Gln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro Pro ThrGly Leu 65 70 75 80 Gln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly ProGlu Lys Leu 85 90 95 Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro ThrGln Val Lys 100 105 110 His Met Met Gln Val Ile Glu Pro Gln Phe Gln ArgAsp Phe Ile Ser 115 120 125 Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val LeuSer Phe Leu Glu Pro 130 135 140 Lys Asp Leu Leu Gln Ala Ala Gln Thr CysArg Tyr Trp Arg Ile Leu 145 150 155 160 Ala Glu Asp Asn Leu Leu Trp ArgGlu Lys Cys Lys Glu Glu Gly Ile 165 170 175 Asp Glu Pro Leu His Ile LysArg Arg Lys Val Ile Lys Pro Gly Phe 180 185 190 Ile His Ser Pro Trp LysSer Ala Tyr Ile Arg Gln His Arg Ile Asp 195 200 205 Thr Asn Trp Arg ArgGly Glu Leu Lys Ser Pro Lys Val Leu Lys Gly 210 215 220 His Asp Asp HisVal Ile Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile 225 230 235 240 Val SerGly Ser Asp Asp Asn Thr Leu Lys Val Trp Ser Ala Val Thr 245 250 255 GlyLys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly Val Trp Ser 260 265 270Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr 275 280285 Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His Thr Leu Tyr 290295 300 Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu Lys Arg Val305 310 315 320 Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp Asp IleGlu Thr 325 330 335 Gly Gln Cys Leu His Val Leu Met Gly His Val Ala AlaVal Arg Cys 340 345 350 Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly AlaTyr Asp Phe Met 355 360 365 Val Lys Val Trp Asp Pro Glu Thr Glu Thr CysLeu His Thr Leu Gln 370 375 380 Gly His Thr Asn Arg Val Tyr Ser Leu GlnPhe Asp Gly Ile His Val 385 390 395 400 Val Ser Gly Ser Leu Asp Thr SerIle Arg Val Trp Asp Val Glu Thr 405 410 415 Gly Asn Cys Ile His Thr LeuThr Gly His Gln Ser Leu Thr Ser Gly 420 425 430 Met Glu Leu Lys Asp AsnIle Leu Val Ser Gly Asn Ala Asp Ser Thr 435 440 445 Val Lys Ile Trp AspIle Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln 450 455 460 Gly Pro Asn LysHis Gln Ser Ala Val Thr Cys Leu Gln Phe Asn Lys 465 470 475 480 Asn PheVal Ile Thr Ser Ser Asp Asp Gly Thr Val Lys Leu Trp Asp 485 490 495 LeuLys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu Glu Ser Gly 500 505 510Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu 515 520525 Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu 530535 540 Val Leu Asp Phe Asp Val Asp Met Lys 545 550 6 545 PRT Homosapiens 6 Met Ile Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser GluVal 1 5 10 15 Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser GluTyr Thr 20 25 30 Ser Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr ThrPhe Gly 35 40 45 Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg ArgIle Thr 50 55 60 Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys MetPhe Gln 65 70 75 80 Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp GluLeu Ile Asp 85 90 95 Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln ValIle Glu Pro 100 105 110 Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro LysGlu Leu Ala Leu 115 120 125 Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp LeuLeu Gln Ala Ala Gln 130 135 140 Thr Cys Arg Tyr Trp Arg Ile Leu Ala GluAsp Asn Leu Leu Trp Arg 145 150 155 160 Glu Lys Cys Lys Glu Glu Gly IleAsp Glu Pro Leu His Ile Lys Arg 165 170 175 Arg Lys Val Ile Lys Pro GlyPhe Ile His Ser Pro Trp Lys Ser Ala 180 185 190 Tyr Ile Arg Gln His ArgIle Asp Thr Asn Trp Arg Arg Gly Glu Leu 195 200 205 Lys Ser Pro Lys ValLeu Lys Gly His Asp Asp His Val Ile Thr Cys 210 215 220 Leu Gln Phe CysGly Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr 225 230 235 240 Leu LysVal Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val 245 250 255 GlyHis Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile 260 265 270Ile Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr 275 280285 Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys 290295 300 Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr305 310 315 320 Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His ValLeu Met 325 330 335 Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp GlyArg Arg Val 340 345 350 Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val TrpAsp Pro Glu Thr 355 360 365 Glu Thr Cys Leu His Thr Leu Gln Gly His ThrAsn Arg Val Tyr Ser 370 375 380 Leu Gln Phe Asp Gly Ile His Val Val SerGly Ser Leu Asp Thr Ser 385 390 395 400 Ile Arg Val Trp Asp Val Glu ThrGly Asn Cys Ile His Thr Leu Thr 405 410 415 Gly His Gln Ser Leu Thr SerGly Met Glu Leu Lys Asp Asn Ile Leu 420 425 430 Val Ser Gly Asn Ala AspSer Thr Val Lys Ile Trp Asp Ile Lys Thr 435 440 445 Gly Gln Cys Leu GlnThr Leu Gln Gly Pro Asn Lys His Gln Ser Ala 450 455 460 Val Thr Cys LeuGln Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp 465 470 475 480 Asp GlyThr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg 485 490 495 AsnLeu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg 500 505 510Ile Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn 515 520525 Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met 530535 540 Lys 545 7 540 PRT Homo sapiens 7 Met Lys Arg Lys Leu Asp His GlySer Glu Val Arg Ser Phe Ser Leu 1 5 10 15 Gly Lys Lys Pro Cys Lys ValSer Glu Tyr Thr Ser Thr Thr Gly Leu 20 25 30 Val Pro Cys Ser Ala Thr ProThr Thr Phe Gly Asp Leu Arg Ala Ala 35 40 45 Asn Gly Gln Gly Gln Gln ArgArg Arg Ile Thr Ser Val Gln Pro Pro 50 55 60 Thr Gly Leu Gln Glu Trp LeuLys Met Phe Gln Ser Trp Ser Gly Pro 65 70 75 80 Glu Lys Leu Leu Ala LeuAsp Glu Leu Ile Asp Ser Cys Glu Pro Thr 85 90 95 Gln Val Lys His Met MetGln Val Ile Glu Pro Gln Phe Gln Arg Asp 100 105 110 Phe Ile Ser Leu LeuPro Lys Glu Leu Ala Leu Tyr Val Leu Ser Phe 115 120 125 Leu Glu Pro LysAsp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr Trp 130 135 140 Arg Ile LeuAla Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys Glu 145 150 155 160 GluGly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys Val Ile Lys 165 170 175Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr Ile Arg Gln His 180 185190 Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val 195200 205 Leu Lys Gly His Asp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly210 215 220 Asn Arg Ile Val Ser Gly Ser Asp Asp Asn Thr Leu Lys Val TrpSer 225 230 235 240 Ala Val Thr Gly Lys Cys Leu Arg Thr Leu Val Gly HisThr Gly Gly 245 250 255 Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile IleSer Gly Ser Thr 260 265 270 Asp Arg Thr Leu Lys Val Trp Asn Ala Glu ThrGly Glu Cys Ile His 275 280 285 Thr Leu Tyr Gly His Thr Ser Thr Val ArgCys Met His Leu His Glu 290 295 300 Lys Arg Val Val Ser Gly Ser Arg AspAla Thr Leu Arg Val Trp Asp 305 310 315 320 Ile Glu Thr Gly Gln Cys LeuHis Val Leu Met Gly His Val Ala Ala 325 330 335 Val Arg Cys Val Gln TyrAsp Gly Arg Arg Val Val Ser Gly Ala Tyr 340 345 350 Asp Phe Met Val LysVal Trp Asp Pro Glu Thr Glu Thr Cys Leu His 355 360 365 Thr Leu Gln GlyHis Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp Gly 370 375 380 Ile His ValVal Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp Asp 385 390 395 400 ValGlu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His Gln Ser Leu 405 410 415Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val Ser Gly Asn Ala 420 425430 Asp Ser Thr Val Lys Ile Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln 435440 445 Thr Leu Gln Gly Pro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln450 455 460 Phe Asn Lys Asn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr ValLys 465 470 475 480 Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn LeuVal Thr Leu 485 490 495 Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg IleArg Ala Ser Asn 500 505 510 Thr Lys Leu Val Cys Ala Val Gly Ser Arg AsnGly Thr Glu Glu Thr 515 520 525 Lys Leu Leu Val Leu Asp Phe Asp Val AspMet Lys 530 535 540 8 589 PRT Homo sapiens 8 Met Ser Lys Pro Gly Lys ProThr Leu Asn His Gly Leu Val Pro Val 1 5 10 15 Asp Leu Lys Ser Ala LysGlu Pro Leu Pro His Gln Thr Val Met Lys 20 25 30 Ile Phe Ser Ile Ser IleIle Ala Gln Gly Leu Pro Phe Cys Arg Arg 35 40 45 Arg Met Lys Arg Lys LeuAsp His Gly Ser Glu Val Arg Ser Phe Ser 50 55 60 Leu Gly Lys Lys Pro CysLys Val Ser Glu Tyr Thr Ser Thr Thr Gly 65 70 75 80 Leu Val Pro Cys SerAla Thr Pro Thr Thr Phe Gly Asp Leu Arg Ala 85 90 95 Ala Asn Gly Gln GlyGln Gln Arg Arg Arg Ile Thr Ser Val Gln Pro 100 105 110 Pro Thr Gly LeuGln Glu Trp Leu Lys Met Phe Gln Ser Trp Ser Gly 115 120 125 Pro Glu LysLeu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys Glu Pro 130 135 140 Thr GlnVal Lys His Met Met Gln Val Ile Glu Pro Gln Phe Gln Arg 145 150 155 160Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val Leu Ser 165 170175 Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln Thr Cys Arg Tyr 180185 190 Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu Trp Arg Glu Lys Cys Lys195 200 205 Glu Glu Gly Ile Asp Glu Pro Leu His Ile Lys Arg Arg Lys ValIle 210 215 220 Lys Pro Gly Phe Ile His Ser Pro Trp Lys Ser Ala Tyr IleArg Gln 225 230 235 240 His Arg Ile Asp Thr Asn Trp Arg Arg Gly Glu LeuLys Ser Pro Lys 245 250 255 Val Leu Lys Gly His Asp Asp His Val Ile ThrCys Leu Gln Phe Cys 260 265 270 Gly Asn Arg Ile Val Ser Gly Ser Asp AspAsn Thr Leu Lys Val Trp 275 280 285 Ser Ala Val Thr Gly Lys Cys Leu ArgThr Leu Val Gly His Thr Gly 290 295 300 Gly Val Trp Ser Ser Gln Met ArgAsp Asn Ile Ile Ile Ser Gly Ser 305 310 315 320 Thr Asp Arg Thr Leu LysVal Trp Asn Ala Glu Thr Gly Glu Cys Ile 325 330 335 His Thr Leu Tyr GlyHis Thr Ser Thr Val Arg Cys Met His Leu His 340 345 350 Glu Lys Arg ValVal Ser Gly Ser Arg Asp Ala Thr Leu Arg Val Trp 355 360 365 Asp Ile GluThr Gly Gln Cys Leu His Val Leu Met Gly His Val Ala 370 375 380 Ala ValArg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser Gly Ala 385 390 395 400Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr Cys Leu 405 410415 His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser Leu Gln Phe Asp 420425 430 Gly Ile His Val Val Ser Gly Ser Leu Asp Thr Ser Ile Arg Val Trp435 440 445 Asp Val Glu Thr Gly Asn Cys Ile His Thr Leu Thr Gly His GlnSer 450 455 460 Leu Thr Ser Gly Met Glu Leu Lys Asp Asn Ile Leu Val SerGly Asn 465 470 475 480 Ala Asp Ser Thr Val Lys Ile Trp Asp Ile Lys ThrGly Gln Cys Leu 485 490 495 Gln Thr Leu Gln Gly Pro Asn Lys His Gln SerAla Val Thr Cys Leu 500 505 510 Gln Phe Asn Lys Asn Phe Val Ile Thr SerSer Asp Asp Gly Thr Val 515 520 525 Lys Leu Trp Asp Leu Lys Thr Gly GluPhe Ile Arg Asn Leu Val Thr 530 535 540 Leu Glu Ser Gly Gly Ser Gly GlyVal Val Trp Arg Ile Arg Ala Ser 545 550 555 560 Asn Thr Lys Leu Val CysAla Val Gly Ser Arg Asn Gly Thr Glu Glu 565 570 575 Thr Lys Leu Leu ValLeu Asp Phe Asp Val Asp Met Lys 580 585 9 559 PRT Homo sapiens 9 Met LysIle Phe Ser Ile Ser Ile Ile Ala Gln Gly Leu Pro Phe Cys 1 5 10 15 ArgArg Arg Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser 20 25 30 PheSer Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser Thr 35 40 45 ThrGly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp Leu 50 55 60 ArgAla Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser Val 65 70 75 80Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser Trp 85 90 95Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp Ser Cys 100 105110 Glu Pro Thr Gln Val Lys His Met Met Gln Val Ile Glu Pro Gln Phe 115120 125 Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala Leu Tyr Val130 135 140 Leu Ser Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala Ala Gln ThrCys 145 150 155 160 Arg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu TrpArg Glu Lys 165 170 175 Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His IleLys Arg Arg Lys 180 185 190 Val Ile Lys Pro Gly Phe Ile His Ser Pro TrpLys Ser Ala Tyr Ile 195 200 205 Arg Gln His Arg Ile Asp Thr Asn Trp ArgArg Gly Glu Leu Lys Ser 210 215 220 Pro Lys Val Leu Lys Gly His Asp AspHis Val Ile Thr Cys Leu Gln 225 230 235 240 Phe Cys Gly Asn Arg Ile ValSer Gly Ser Asp Asp Asn Thr Leu Lys 245 250 255 Val Trp Ser Ala Val ThrGly Lys Cys Leu Arg Thr Leu Val Gly His 260 265 270 Thr Gly Gly Val TrpSer Ser Gln Met Arg Asp Asn Ile Ile Ile Ser 275 280 285 Gly Ser Thr AspArg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu 290 295 300 Cys Ile HisThr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His 305 310 315 320 LeuHis Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg 325 330 335Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly His 340 345350 Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val Val Ser 355360 365 Gly Ala Tyr Asp Phe Met Val Lys Val Trp Asp Pro Glu Thr Glu Thr370 375 380 Cys Leu His Thr Leu Gln Gly His Thr Asn Arg Val Tyr Ser LeuGln 385 390 395 400 Phe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp ThrSer Ile Arg 405 410 415 Val Trp Asp Val Glu Thr Gly Asn Cys Ile His ThrLeu Thr Gly His 420 425 430 Gln Ser Leu Thr Ser Gly Met Glu Leu Lys AspAsn Ile Leu Val Ser 435 440 445 Gly Asn Ala Asp Ser Thr Val Lys Ile TrpAsp Ile Lys Thr Gly Gln 450 455 460 Cys Leu Gln Thr Leu Gln Gly Pro AsnLys His Gln Ser Ala Val Thr 465 470 475 480 Cys Leu Gln Phe Asn Lys AsnPhe Val Ile Thr Ser Ser Asp Asp Gly 485 490 495 Thr Val Lys Leu Trp AspLeu Lys Thr Gly Glu Phe Ile Arg Asn Leu 500 505 510 Val Thr Leu Glu SerGly Gly Ser Gly Gly Val Val Trp Arg Ile Arg 515 520 525 Ala Ser Asn ThrLys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr 530 535 540 Glu Glu ThrLys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 545 550 555 10 540 PRTHomo sapiens 10 Met Lys Arg Lys Leu Asp His Gly Ser Glu Val Arg Ser PheSer Leu 1 5 10 15 Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr Thr Ser ThrThr Gly Leu 20 25 30 Val Pro Cys Ser Ala Thr Pro Thr Thr Phe Gly Asp LeuArg Ala Ala 35 40 45 Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile Thr Ser ValGln Pro Pro 50 55 60 Thr Gly Leu Gln Glu Trp Leu Lys Met Phe Gln Ser TrpSer Gly Pro 65 70 75 80 Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile Asp SerCys Glu Pro Thr 85 90 95 Gln Val Lys His Met Met Gln Val Ile Glu Pro GlnPhe Gln Arg Asp 100 105 110 Phe Ile Ser Leu Leu Pro Lys Glu Leu Ala LeuTyr Val Leu Ser Phe 115 120 125 Leu Glu Pro Lys Asp Leu Leu Gln Ala AlaGln Thr Cys Arg Tyr Trp 130 135 140 Arg Ile Leu Ala Glu Asp Asn Leu LeuTrp Arg Glu Lys Cys Lys Glu 145 150 155 160 Glu Gly Ile Asp Glu Pro LeuHis Ile Lys Arg Arg Lys Val Ile Lys 165 170 175 Pro Gly Phe Ile His SerPro Trp Lys Ser Ala Tyr Ile Arg Gln His 180 185 190 Arg Ile Asp Thr AsnTrp Arg Arg Gly Glu Leu Lys Ser Pro Lys Val 195 200 205 Leu Lys Gly HisAsp Asp His Val Ile Thr Cys Leu Gln Phe Cys Gly 210 215 220 Asn Arg IleVal Ser Gly Ser Asp Asp Asn Thr Leu Lys Val Trp Ser 225 230 235 240 AlaVal Thr Gly Lys Cys Leu Arg Thr Leu Val Gly His Thr Gly Gly 245 250 255Val Trp Ser Ser Gln Met Arg Asp Asn Ile Ile Ile Ser Gly Ser Thr 260 265270 Asp Arg Thr Leu Lys Val Trp Asn Ala Glu Thr Gly Glu Cys Ile His 275280 285 Thr Leu Tyr Gly His Thr Ser Thr Val Arg Cys Met His Leu His Glu290 295 300 Lys Arg Val Val Ser Gly Ser Arg Asp Ala Thr Leu Arg Val TrpAsp 305 310 315 320 Ile Glu Thr Gly Gln Cys Leu His Val Leu Met Gly HisVal Ala Ala 325 330 335 Val Arg Cys Val Gln Tyr Asp Gly Arg Arg Val ValSer Gly Ala Tyr 340 345 350 Asp Phe Met Val Lys Val Trp Asp Pro Glu ThrGlu Thr Cys Leu His 355 360 365 Thr Leu Gln Gly His Thr Asn Arg Val TyrSer Leu Gln Phe Asp Gly 370 375 380 Ile His Val Val Ser Gly Ser Leu AspThr Ser Ile Arg Val Trp Asp 385 390 395 400 Val Glu Thr Gly Asn Cys IleHis Thr Leu Thr Gly His Gln Ser Leu 405 410 415 Thr Ser Gly Met Glu LeuLys Asp Asn Ile Leu Val Ser Gly Asn Ala 420 425 430 Asp Ser Thr Val LysIle Trp Asp Ile Lys Thr Gly Gln Cys Leu Gln 435 440 445 Thr Leu Gln GlyPro Asn Lys His Gln Ser Ala Val Thr Cys Leu Gln 450 455 460 Phe Asn LysAsn Phe Val Ile Thr Ser Ser Asp Asp Gly Thr Val Lys 465 470 475 480 LeuTrp Asp Leu Lys Thr Gly Glu Phe Ile Arg Asn Leu Val Thr Leu 485 490 495Glu Ser Gly Gly Ser Gly Gly Val Val Trp Arg Ile Arg Ala Ser Asn 500 505510 Thr Lys Leu Val Cys Ala Val Gly Ser Arg Asn Gly Thr Glu Glu Thr 515520 525 Lys Leu Leu Val Leu Asp Phe Asp Val Asp Met Lys 530 535 540 1134 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 11 cgggatccac catggatgat ggatcgatga cacc 34 12 33DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 12 ggaattcctt aagggtatac agcatcaaag tcg 33 13 25DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 13 tcacttcatg tccacatcaa agtcc 25 14 26 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer 14 ggtaattaca agttcttgtt gaactg 26 15 22 DNA Artificial SequenceDescription of Artificial Sequence Oligonucleotide primer 15 ccctgcaacgtgtgtagaca gg 22 16 24 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer 16 ccagtctctg cattccacac tttg 24 17 23DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 17 ctcagacagg tcaggacatt tgg 23 18 33 DNAArtificial Sequence Description of Artificial Sequence Oligonucleotideprimer 18 ggaattccat gaaaagattg gaccatggtt ctg 33 19 34 DNA ArtificialSequence Description of Artificial Sequence Oligonucleotide primer 19ggaattcctc acttcatgtc acatcaaagt ccag 34 20 1881 DNA Artificial SequenceDescription of Artificial Sequence 6 myc tagged homo sapiens 20atggagcaaa agctcatttc tgaagaggac ttgaatgaaa tggagcaaaa gctcatttct 60gaagaggact tgaatgaaat ggagcaaaag ctcatttctg aagaggactt gaatgaaatg 120gagcaaaagc tcatttctga agaggacttg aatgaaatgg agcaaaagct catttctgaa 180gaggacttga atgaaatgga gagcttgggc gacctcacca tggagcaaaa gctcatttct 240gaagaggact tgaattccat gaaaagaaag ttggaccatg gttctgaggt ccgctctttt 300tctttgggaa agaaaccatg caaagtctca gaatatacaa gtaccactgg gcttgtacca 360tgttcagcaa caccaacaac ttttggggac ctcagagcag ccaatggcca agggcaacaa 420cgacgccgaa ttacatctgt ccagccacct acaggcctcc aggaatggct aaaaatgttt 480cagagctgga gtggaccaga gaaattgctt gctttagatg aactcattga tagttgtgaa 540ccaacacaag taaaacatat gatgcaagtg atagaacccc agtttcaacg agacttcatt 600tcattgctcc ctaaagagtt ggcactctat gtgctttcat tcctggaacc caaagacctg 660ctacaagcag ctcagacatg tcgctactgg agaattttgg ctgaagacaa ccttctctgg 720agagagaaat gcaaagaaga ggggattgat gaaccattgc acatcaagag aagaaaagta 780ataaaaccag gtttcataca cagtccatgg aaaagtgcat acatcagaca gcacagaatt 840gatactaact ggaggcgagg agaactcaaa tctcctaagg tgctgaaagg acatgatgat 900catgtgatca catgcttaca gttttgtggt aaccgaatag ttagtggttc tgatgacaac 960actttaaaag tttggtcagc agtcacaggc aaatgtctga gaacattagt gggacataca 1020ggtggagtat ggtcatcaca aatgagggac aacatcatca ttagtggatc tacagatcgg 1080acactcaaag tgtggaatgc agagactgga gaatgtatac acaccttata tgggcatact 1140tccactgtgc gttgtatgca tcttcatgaa aaaagagttg ttagcggttc tcgagatgcc 1200actcttaggg tttgggatat tgagacaggc cagtgtttac atgttttgat gggtcatgtt 1260gcagcagtcc gctgtgttca atatgatggc aggagggttg ttagtggagc atatgatttt 1320atggtaaagg tgtgggatcc agagactgaa acctgtctac acacgttgca ggggcatact 1380aatagagtct attcattaca gtttgatggt atccatgtgg tgagtggatc tcttgataca 1440tccatccgtg tttgggatgt ggagacaggg aattgcattc acacgttaac agggcaccag 1500tcgttaacaa gtggaatgga actcaaagac aatattcttg tctctgggaa tgcagattct 1560acagttaaaa tctgggatat caaaacagga cagtgtttac aaacattgca aggtcccaac 1620aagcatcaga gtgctgtgac ctgtttacag ttcaacaaga actttgtaat taccagctca 1680gatgatggaa ctgtaaaact atgggacttg aaaacgggtg aatttattcg aaacctagtc 1740acattggaga gtggggggag tgggggagtt gtgtggcgga tcagagcctc aaacacaaag 1800ctggtgtgtg cagttgggag tcggaatggg actgaagaaa ccaagctgct ggtgctggac 1860tttgatgtgg acatgaagtg a 1881 21 626 PRT Artificial Sequence Descriptionof Artificial Sequence 6 myc tagged homo sapien 21 Met Glu Gln Lys LeuIle Ser Glu Glu Asp Leu Asn Glu Met Glu Gln 1 5 10 15 Lys Leu Ile SerGlu Glu Asp Leu Asn Glu Met Glu Gln Lys Leu Ile 20 25 30 Ser Glu Glu AspLeu Asn Glu Met Glu Gln Lys Leu Ile Ser Glu Glu 35 40 45 Asp Leu Asn GluMet Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn 50 55 60 Glu Met Glu SerLeu Gly Asp Leu Thr Met Glu Gln Lys Leu Ile Ser 65 70 75 80 Glu Glu AspLeu Asn Ser Met Lys Arg Lys Leu Asp His Gly Ser Glu 85 90 95 Val Arg SerPhe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu Tyr 100 105 110 Thr SerThr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr Phe 115 120 125 GlyAsp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg Ile 130 135 140Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met Phe 145 150155 160 Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu Ile165 170 175 Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln Val IleGlu 180 185 190 Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro Lys GluLeu Ala 195 200 205 Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp Leu LeuGln Ala Ala 210 215 220 Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala Glu AspAsn Leu Leu Trp 225 230 235 240 Arg Glu Lys Cys Lys Glu Glu Gly Ile AspGlu Pro Leu His Ile Lys 245 250 255 Arg Arg Lys Val Ile Lys Pro Gly PheIle His Ser Pro Trp Lys Ser 260 265 270 Ala Tyr Ile Arg Gln His Arg IleAsp Thr Asn Trp Arg Arg Gly Glu 275 280 285 Leu Lys Ser Pro Lys Val LeuLys Gly His Asp Asp His Val Ile Thr 290 295 300 Cys Leu Gln Phe Cys GlyAsn Arg Ile Val Ser Gly Ser Asp Asp Asn 305 310 315 320 Thr Leu Lys ValTrp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr Leu 325 330 335 Val Gly HisThr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn Ile 340 345 350 Ile IleSer Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala Glu 355 360 365 ThrGly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val Arg 370 375 380Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp Ala 385 390395 400 Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val Leu405 410 415 Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp Gly ArgArg 420 425 430 Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val Trp AspPro Glu 435 440 445 Thr Glu Thr Cys Leu His Thr Leu Gln Gly His Thr AsnArg Val Tyr 450 455 460 Ser Leu Gln Phe Asp Gly Ile His Val Val Ser GlySer Leu Asp Thr 465 470 475 480 Ser Ile Arg Val Trp Asp Val Glu Thr GlyAsn Cys Ile His Thr Leu 485 490 495 Thr Gly His Gln Ser Leu Thr Ser GlyMet Glu Leu Lys Asp Asn Ile 500 505 510 Leu Val Ser Gly Asn Ala Asp SerThr Val Lys Ile Trp Asp Ile Lys 515 520 525 Thr Gly Gln Cys Leu Gln ThrLeu Gln Gly Pro Asn Lys His Gln Ser 530 535 540 Ala Val Thr Cys Leu GlnPhe Asn Lys Asn Phe Val Ile Thr Ser Ser 545 550 555 560 Asp Asp Gly ThrVal Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe Ile 565 570 575 Arg Asn LeuVal Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val Trp 580 585 590 Arg IleArg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser Arg 595 600 605 AsnGly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val Asp 610 615 620Met Lys 625 22 31 DNA Artificial Sequence Description of ArtificialSequence Oligonucleotide primer 22 gggtacccct cattattccc tcgagttctt c 3123 29 DNA Artificial Sequence Description of Artificial SequenceOligonucleotide primer 23 ggaattcctt catgtccaca tcaaagtcc 29 24 2010 DNAArtificial Sequence Description of Artificial Sequence V5HIS tagged homosapien 24 atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactgtggagttttg 60 ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtccacattagaa 120 tctgtgacat acctacctga aaaaggttta tattgtcaga gactgccaagcagccggaca 180 cacgggggca cagaatcact gaaggggaaa aatacagaaa atatgggtttctacggcaca 240 ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctgaggtccgctct 300 ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccactgggcttgta 360 ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatggccaagggcaa 420 caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatggctaaaaatg 480 tttcagagct ggagtggacc agagaaattg cttgctttag atgaactcattgatagttgt 540 gaaccaacac aagtaaaaca tatgatgcaa gtgatagaac cccagtttcaacgagacttc 600 atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctggaacccaaagac 660 ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaagacaaccttctc 720 tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaagagaagaaaa 780 gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcagacagcacaga 840 attgatacta actggaggcg aggagaactc aaatctccta aggtgctgaaaggacatgat 900 gatcatgtga tcacatgctt acagttttgt ggtaaccgaa tagttagtggttctgatgac 960 aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacattagtgggacat 1020 acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtggatctacagat 1080 cggacactca aagtgtggaa tgcagagact ggagaatgta tacacaccttatatgggcat 1140 acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcggttctcgagat 1200 gccactctta gggtttggga tattgagaca ggccagtgtt tacatgttttgatgggtcat 1260 gttgcagcag tccgctgtgt tcaatatgat ggcaggaggg ttgttagtggagcatatgat 1320 tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgttgcaggggcat 1380 actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtggatctcttgat 1440 acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgttaacagggcac 1500 cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgggaatgcagat 1560 tctacagtta aaatctggga tatcaaaaca ggacagtgtt tacaaacattgcaaggtccc 1620 aacaagcatc agagtgctgt gacctgttta cagttcaaca agaactttgtaattaccagc 1680 tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttattcgaaaccta 1740 gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagcctcaaacaca 1800 aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagctgctggtgctg 1860 gactttgatg tggacatgaa ggaattctgc agatatccag cacagtggcggccgctcgag 1920 tctagagggc ccttcgaagg taagcctatc cctaaccctc tcctcggtctcgattctacg 1980 cgtaccggtc atcatcacca tcaccattga 2010 25 669 PRTArtificial Sequence Description of Artificial Sequence V5HIS tagged homosapien 25 Met Cys Val Pro Arg Ser Gly Leu Ile Leu Ser Cys Ile Cys LeuTyr 1 5 10 15 Cys Gly Val Leu Leu Pro Val Leu Leu Pro Asn Leu Pro PheLeu Thr 20 25 30 Cys Leu Ser Met Ser Thr Leu Glu Ser Val Thr Tyr Leu ProGlu Lys 35 40 45 Gly Leu Tyr Cys Gln Arg Leu Pro Ser Ser Arg Thr His GlyGly Thr 50 55 60 Glu Ser Leu Lys Gly Lys Asn Thr Glu Asn Met Gly Phe TyrGly Thr 65 70 75 80 Leu Lys Met Ile Phe Tyr Lys Met Lys Arg Lys Leu AspHis Gly Ser 85 90 95 Glu Val Arg Ser Phe Ser Leu Gly Lys Lys Pro Cys LysVal Ser Glu 100 105 110 Tyr Thr Ser Thr Thr Gly Leu Val Pro Cys Ser AlaThr Pro Thr Thr 115 120 125 Phe Gly Asp Leu Arg Ala Ala Asn Gly Gln GlyGln Gln Arg Arg Arg 130 135 140 Ile Thr Ser Val Gln Pro Pro Thr Gly LeuGln Glu Trp Leu Lys Met 145 150 155 160 Phe Gln Ser Trp Ser Gly Pro GluLys Leu Leu Ala Leu Asp Glu Leu 165 170 175 Ile Asp Ser Cys Glu Pro ThrGln Val Lys His Met Met Gln Val Ile 180 185 190 Glu Pro Gln Phe Gln ArgAsp Phe Ile Ser Leu Leu Pro Lys Glu Leu 195 200 205 Ala Leu Tyr Val LeuSer Phe Leu Glu Pro Lys Asp Leu Leu Gln Ala 210 215 220 Ala Gln Thr CysArg Tyr Trp Arg Ile Leu Ala Glu Asp Asn Leu Leu 225 230 235 240 Trp ArgGlu Lys Cys Lys Glu Glu Gly Ile Asp Glu Pro Leu His Ile 245 250 255 LysArg Arg Lys Val Ile Lys Pro Gly Phe Ile His Ser Pro Trp Lys 260 265 270Ser Ala Tyr Ile Arg Gln His Arg Ile Asp Thr Asn Trp Arg Arg Gly 275 280285 Glu Leu Lys Ser Pro Lys Val Leu Lys Gly His Asp Asp His Val Ile 290295 300 Thr Cys Leu Gln Phe Cys Gly Asn Arg Ile Val Ser Gly Ser Asp Asp305 310 315 320 Asn Thr Leu Lys Val Trp Ser Ala Val Thr Gly Lys Cys LeuArg Thr 325 330 335 Leu Val Gly His Thr Gly Gly Val Trp Ser Ser Gln MetArg Asp Asn 340 345 350 Ile Ile Ile Ser Gly Ser Thr Asp Arg Thr Leu LysVal Trp Asn Ala 355 360 365 Glu Thr Gly Glu Cys Ile His Thr Leu Tyr GlyHis Thr Ser Thr Val 370 375 380 Arg Cys Met His Leu His Glu Lys Arg ValVal Ser Gly Ser Arg Asp 385 390 395 400 Ala Thr Leu Arg Val Trp Asp IleGlu Thr Gly Gln Cys Leu His Val 405 410 415 Leu Met Gly His Val Ala AlaVal Arg Cys Val Gln Tyr Asp Gly Arg 420 425 430 Arg Val Val Ser Gly AlaTyr Asp Phe Met Val Lys Val Trp Asp Pro 435 440 445 Glu Thr Glu Thr CysLeu His Thr Leu Gln Gly His Thr Asn Arg Val 450 455 460 Tyr Ser Leu GlnPhe Asp Gly Ile His Val Val Ser Gly Ser Leu Asp 465 470 475 480 Thr SerIle Arg Val Trp Asp Val Glu Thr Gly Asn Cys Ile His Thr 485 490 495 LeuThr Gly His Gln Ser Leu Thr Ser Gly Met Glu Leu Lys Asp Asn 500 505 510Ile Leu Val Ser Gly Asn Ala Asp Ser Thr Val Lys Ile Trp Asp Ile 515 520525 Lys Thr Gly Gln Cys Leu Gln Thr Leu Gln Gly Pro Asn Lys His Gln 530535 540 Ser Ala Val Thr Cys Leu Gln Phe Asn Lys Asn Phe Val Ile Thr Ser545 550 555 560 Ser Asp Asp Gly Thr Val Lys Leu Trp Asp Leu Lys Thr GlyGlu Phe 565 570 575 Ile Arg Asn Leu Val Thr Leu Glu Ser Gly Gly Ser GlyGly Val Val 580 585 590 Trp Arg Ile Arg Ala Ser Asn Thr Lys Leu Val CysAla Val Gly Ser 595 600 605 Arg Asn Gly Thr Glu Glu Thr Lys Leu Leu ValLeu Asp Phe Asp Val 610 615 620 Asp Met Lys Glu Phe Cys Arg Tyr Pro AlaGln Trp Arg Pro Leu Glu 625 630 635 640 Ser Arg Gly Pro Phe Glu Gly LysPro Ile Pro Asn Pro Leu Leu Gly 645 650 655 Leu Asp Ser Thr Arg Thr GlyHis His His His His His 660 665 26 2001 DNA Artificial SequenceDescription of Artificial Sequence MYCHIS tagged homo sapiens 26atgtgtgtcc cgagaagcgg tttgatactg agctgcattt gcctttactg tggagttttg 60ttgccggttc tgctccctaa tcttcctttt ctgacgtgcc tgagcatgtc cacattagaa 120tctgtgacat acctacctga aaaaggttta tattgtcaga gactgccaag cagccggaca 180cacgggggca cagaatcact gaaggggaaa aatacagaaa atatgggttt ctacggcaca 240ttaaaaatga ttttttacaa aatgaaaaga aagttggacc atggttctga ggtccgctct 300ttttctttgg gaaagaaacc atgcaaagtc tcagaatata caagtaccac tgggcttgta 360ccatgttcag caacaccaac aacttttggg gacctcagag cagccaatgg ccaagggcaa 420caacgacgcc gaattacatc tgtccagcca cctacaggcc tccaggaatg gctaaaaatg 480tttcagagct ggagtggacc agagaaattg cttgctttag atgaactcat tgatagttgt 540gaaccaacac aagtaaaaca tatgatgcaa gtgatagaac cccagtttca acgagacttc 600atttcattgc tccctaaaga gttggcactc tatgtgcttt cattcctgga acccaaagac 660ctgctacaag cagctcagac atgtcgctac tggagaattt tggctgaaga caaccttctc 720tggagagaga aatgcaaaga agaggggatt gatgaaccat tgcacatcaa gagaagaaaa 780gtaataaaac caggtttcat acacagtcca tggaaaagtg catacatcag acagcacaga 840attgatacta actggaggcg aggagaactc aaatctccta aggtgctgaa aggacatgat 900gatcatgtga tcacatgctt acagttttgt ggtaaccgaa tagttagtgg ttctgatgac 960aacactttaa aagtttggtc agcagtcaca ggcaaatgtc tgagaacatt agtgggacat 1020acaggtggag tatggtcatc acaaatgaga gacaacatca tcattagtgg atctacagat 1080cggacactca aagtgtggaa tgcagagact ggagaatgta tacacacctt atatgggcat 1140acttccactg tgcgttgtat gcatcttcat gaaaaaagag ttgttagcgg ttctcgagat 1200gccactctta gggtttggga tattgagaca ggccagtgtt tacatgtttt gatgggtcat 1260gttgcagcag tccgctgtgt tcaatatgat ggcaggaggg ttgttagtgg agcatatgat 1320tttatggtaa aggtgtggga tccagagact gaaacctgtc tacacacgtt gcaggggcat 1380actaatagag tctattcatt acagtttgat ggtatccatg tggtgagtgg atctcttgat 1440acatcaatcc gtgtttggga tgtggagaca gggaattgca ttcacacgtt aacagggcac 1500cagtcgttaa caagtggaat ggaactcaaa gacaatattc ttgtctctgg gaatgcagat 1560tctacagtta aaatctggga tatcaaaaca ggacagtgtt tacaaacatt gcaaggtccc 1620aacaagcatc agagtgctgt gacctgttta cagttcaaca agaactttgt aattaccagc 1680tcagatgatg gaactgtaaa actatgggac ttgaaaacgg gtgaatttat tcgaaaccta 1740gtcacattgg agagtggggg gagtggggga gttgtgtggc ggatcagagc ctcaaacaca 1800aagctggtgt gtgcagttgg gagtcggaat gggactgaag aaaccaagct gctggtgctg 1860gactttgatg tggacatgaa ggaattctgc agatatccag cacagtggcg gccgctcgag 1920tctagagggc ccttcgaaca aaaactcatc tcagaagagg atctgaatat gcataccggt 1980catcatcacc atcaccattg a 2001 27 666 PRT Artificial Sequence Descriptionof Artificial Sequence MYCHIS tagged homo sapiens 27 Met Cys Val Pro ArgSer Gly Leu Ile Leu Ser Cys Ile Cys Leu Tyr 1 5 10 15 Cys Gly Val LeuLeu Pro Val Leu Leu Pro Asn Leu Pro Phe Leu Thr 20 25 30 Cys Leu Ser MetSer Thr Leu Glu Ser Val Thr Tyr Leu Pro Glu Lys 35 40 45 Gly Leu Tyr CysGln Arg Leu Pro Ser Ser Arg Thr His Gly Gly Thr 50 55 60 Glu Ser Leu LysGly Lys Asn Thr Glu Asn Met Gly Phe Tyr Gly Thr 65 70 75 80 Leu Lys MetIle Phe Tyr Lys Met Lys Arg Lys Leu Asp His Gly Ser 85 90 95 Glu Val ArgSer Phe Ser Leu Gly Lys Lys Pro Cys Lys Val Ser Glu 100 105 110 Tyr ThrSer Thr Thr Gly Leu Val Pro Cys Ser Ala Thr Pro Thr Thr 115 120 125 PheGly Asp Leu Arg Ala Ala Asn Gly Gln Gly Gln Gln Arg Arg Arg 130 135 140Ile Thr Ser Val Gln Pro Pro Thr Gly Leu Gln Glu Trp Leu Lys Met 145 150155 160 Phe Gln Ser Trp Ser Gly Pro Glu Lys Leu Leu Ala Leu Asp Glu Leu165 170 175 Ile Asp Ser Cys Glu Pro Thr Gln Val Lys His Met Met Gln ValIle 180 185 190 Glu Pro Gln Phe Gln Arg Asp Phe Ile Ser Leu Leu Pro LysGlu Leu 195 200 205 Ala Leu Tyr Val Leu Ser Phe Leu Glu Pro Lys Asp LeuLeu Gln Ala 210 215 220 Ala Gln Thr Cys Arg Tyr Trp Arg Ile Leu Ala GluAsp Asn Leu Leu 225 230 235 240 Trp Arg Glu Lys Cys Lys Glu Glu Gly IleAsp Glu Pro Leu His Ile 245 250 255 Lys Arg Arg Lys Val Ile Lys Pro GlyPhe Ile His Ser Pro Trp Lys 260 265 270 Ser Ala Tyr Ile Arg Gln His ArgIle Asp Thr Asn Trp Arg Arg Gly 275 280 285 Glu Leu Lys Ser Pro Lys ValLeu Lys Gly His Asp Asp His Val Ile 290 295 300 Thr Cys Leu Gln Phe CysGly Asn Arg Ile Val Ser Gly Ser Asp Asp 305 310 315 320 Asn Thr Leu LysVal Trp Ser Ala Val Thr Gly Lys Cys Leu Arg Thr 325 330 335 Leu Val GlyHis Thr Gly Gly Val Trp Ser Ser Gln Met Arg Asp Asn 340 345 350 Ile IleIle Ser Gly Ser Thr Asp Arg Thr Leu Lys Val Trp Asn Ala 355 360 365 GluThr Gly Glu Cys Ile His Thr Leu Tyr Gly His Thr Ser Thr Val 370 375 380Arg Cys Met His Leu His Glu Lys Arg Val Val Ser Gly Ser Arg Asp 385 390395 400 Ala Thr Leu Arg Val Trp Asp Ile Glu Thr Gly Gln Cys Leu His Val405 410 415 Leu Met Gly His Val Ala Ala Val Arg Cys Val Gln Tyr Asp GlyArg 420 425 430 Arg Val Val Ser Gly Ala Tyr Asp Phe Met Val Lys Val TrpAsp Pro 435 440 445 Glu Thr Glu Thr Cys Leu His Thr Leu Gln Gly His ThrAsn Arg Val 450 455 460 Tyr Ser Leu Gln Phe Asp Gly Ile His Val Val SerGly Ser Leu Asp 465 470 475 480 Thr Ser Ile Arg Val Trp Asp Val Glu ThrGly Asn Cys Ile His Thr 485 490 495 Leu Thr Gly His Gln Ser Leu Thr SerGly Met Glu Leu Lys Asp Asn 500 505 510 Ile Leu Val Ser Gly Asn Ala AspSer Thr Val Lys Ile Trp Asp Ile 515 520 525 Lys Thr Gly Gln Cys Leu GlnThr Leu Gln Gly Pro Asn Lys His Gln 530 535 540 Ser Ala Val Thr Cys LeuGln Phe Asn Lys Asn Phe Val Ile Thr Ser 545 550 555 560 Ser Asp Asp GlyThr Val Lys Leu Trp Asp Leu Lys Thr Gly Glu Phe 565 570 575 Ile Arg AsnLeu Val Thr Leu Glu Ser Gly Gly Ser Gly Gly Val Val 580 585 590 Trp ArgIle Arg Ala Ser Asn Thr Lys Leu Val Cys Ala Val Gly Ser 595 600 605 ArgAsn Gly Thr Glu Glu Thr Lys Leu Leu Val Leu Asp Phe Asp Val 610 615 620Asp Met Lys Glu Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Leu Glu 625 630635 640 Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn645 650 655 Met His Thr Gly His His His His His His 660 665

What is claimed is:
 1. An isolated nucleic acid molecule comprising apolynucleotide having a sequence at least 95% identical to a sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding a human sel-10 polypeptide having the complete amino acidsequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4,SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or as encoded by the cDNAclone contained in ATCC Deposit No.98978; (b) a nucleotide sequenceencoding a human sel-10 polypeptide having the complete amino acidsequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:9,and SEQ ID NO:10, or as encoded by the cDNA clone contained in ATCCDeposit No. 98979; and (c) a nucleotide sequence complementary to thenucleotide sequence of (a) or (b).
 2. An isolated nucleic acid moleculecomprising polynucleotide which hybridizes under stringent conditions toa polynucleotide having the nucleotide sequence in (a), (b), or (c) ofclaim
 1. 3. The nucleic acid molecule of claim 1, wherein saidpolynucleotide of 1(a) encodes a human sel-10 polypeptide having thecomplete amino acid sequence of SEQ ID NO:3.
 4. The nucleic acidmolecule of claim 3, wherein said polynucleotide molecule of 1(a)comprises the nucleotide sequence of residues 45-1928 of SEQ ID NO:1. 5.The nucleic acid molecule of claim 1, wherein said polynucleotide of1(a) encodes a human sel-10 polypeptide having the complete amino acidsequence of SEQ ID NO:4.
 6. The nucleic acid molecule of claim 5,wherein said polynucleotide molecule of 1(a) comprises the nucleotidesequence of residues 150-1928 of SEQ ID NO:1.
 7. The nucleic acidmolecule of claim 1, wherein said polynucleotide of 1(a) encodes a humansel-10 polypeptide having the complete amino acid sequence of SEQ IDNO:5.
 8. The nucleic acid molecule of claim 7, wherein saidpolynucleotide molecule of 1(a) comprises the nucleotide sequence ofresidues 267-1928 of SEQ ID NO:1.
 9. The nucleic acid molecule of claim1, wherein said polynucleotide of 1(a) encodes a human sel-10polypeptide having the complete amino acid sequence of SEQ ID NO:6. 10.The nucleic acid molecule of claim 9, wherein said polynucleotidemolecule of 1(a) comprises the nucleotide sequence of residues 291-1928of SEQ ID NO:1.
 11. The nucleic acid molecule of claim 1, wherein saidpolynucleotide of 1(a) encodes a human sel-10 polypeptide having thecomplete amino acid sequence of SEQ ID NO:7.
 12. The nucleic acidmolecule of claim 11, wherein said polynucleotide molecule of 1 (a)comprises the nucleotide sequence of residues 306-1928 of SEQ ID NO:1.13. The nucleic acid molecule of claim 1, wherein said polynucleotide of1(b) encodes a human sel-10 polypeptide having the complete amino acidsequence of SEQ ID NO:8.
 14. The nucleic acid molecule of claim 13wherein said polynucleotide molecule of 1(b) comprises the nucleotidesequence of residues 180-1949 of SEQ ID NO:2.
 15. The nucleic acidmolecule of claim 1, wherein said polynucleotide of 1(b) encodes a humansel-10 polypeptide having the complete amino acid sequence of SEQ IDNO:9.
 16. The nucleic acid molecule of claim 15, wherein saidpolynucleotide molecule of 1(b) comprises the nucleotide sequence ofresidues 270-1949 of SEQ ID NO:2.
 17. The nucleic acid molecule of claim1, wherein said polynucleotide of 1(b) encodes a human sel-10polypeptide having the complete amino acid sequence of SEQ ID NO:10. 18.The nucleic acid molecule of claim 17, wherein said polynucleotidemolecule of 1(b) comprises the nucleotide sequence of residues 327-1949of SEQ ID NO:2.
 19. A vector comprising the nucleic acid molecule ofclaim
 1. 20. The vector of claim 19, wherein said nucleic acid moleculeof claim 1 is operably linked to a promoter for the expression of asel-10 polypeptide.
 21. A host cell comprising the vector of claim 19.22. The host cell of claim 21, wherein said host is a eukaryotic host.23. A method of obtaining a sel-10 polypeptide comprising culturing thehost cell of claim 22 and isolating said sel-10 polypeptide.
 24. Anisolated sel-10 polypeptide comprising (a) an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, and SEQ ID NO:7, or as encoded by the cDNA clonecontained in ATCC Deposit No. 98978; (b) an amino acid sequence selectedfrom the group consisting of SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10,or as encoded by the cDNA clone contained in ATCC Deposit No.
 98979. 25.The isolated sel-10 polypeptide of claim 24, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:3.
 26. The isolatedsel-10 polypeptide of claim 24, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:4.
 27. The isolated sel-10 polypeptideof claim 24, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:5.
 28. The isolated sel-10 polypeptide of claim 24, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:6. 29.The isolated sel-10 polypeptide of claim 24, wherein said polypeptidecomprises the amino acid sequence of SEQ ID NO:7.
 30. The isolatedsel-10 polypeptide of claim 24, wherein said polypeptide comprises theamino acid sequence of SEQ ID NO:8.
 31. The isolated sel-10 polypeptideof claim 24, wherein said polypeptide comprises the amino acid sequenceof SEQ ID NO:9.
 32. The isolated sel-10 polypeptide of claim 24, whereinsaid polypeptide comprises the amino acid sequence of SEQ ID NO:10. 33.An isolated antibody that binds specifically to the sel-10 polypeptideof claim
 24. 34. A cell line having altered Aβ processing that expressesany of the sel-10 isolated nucleic acid molecules of claim
 1. 35. Thecell line of claim 34, wherein said Aβ processing is increased.
 36. Thecell line of claim 34, wherein said Aβ processing is decreased.
 37. Thecell line of claim 34, wherein said cell line is 6myc-N-sel 10/2. 38.The cell line of claim 34, wherein said cell line is 6myc-N-sel 10/6.39. A method for the identification of an agent capable of altering theratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ produced in any of the cell lines ofclaims 34, 37, and 38, comprising the steps of: (a) obtaining a testculture and a control culture of said cell line; (b) contacting saidtest culture with a test agent; (c) measuring the levels of Aβ₁₋₄₀ andAβ₁₋₄₂ produced by said test culture of step (b) and said controlculture; (d) calculating the ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ for said testculture and said control culture from the levels of Aβ₁₋₄₀ and Aβ₁₋₄₂measured in step (c); and (e) comparing the ratio ofAβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ measured for said test culture and said controlculture in step (d); whereby a determination that the ratio ofAβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ for said test culture is higher or lower than ratioof Aβ₁₋₄₀/A₁₋₄₀+Aβ₁₋₄₂ for said control culture indicates that said testagent has altered the ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂.
 40. The method ofclaim 39, wherein said ratio of Aβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ is increased bysaid test agent.
 41. The method of claim 39, wherein said ratio ofAβ₁₋₄₀/Aβ₁₋₄₀+Aβ₁₋₄₂ is decreased by said test agent.